Human tissue inhibitor of metalloproteinase-4

ABSTRACT

A human tissue inhibitor of metalloproteinases-4 polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques. Also disclosed are methods for utilizing such polypeptide for the treatment of diseases, including arthritis and cancer. Antagonists against such polypeptides and their use as a therapeutic to resorb scar tissue are also disclosed. Diagnostic assays for detecting levels of human TIMP-4 protein and mutations in human TIMP-4 nucleic acid sequence are also disclosed.

This application is a Continuation-in-Part of U.S. application Ser. No.09/387,525, filed Sep. 1, 1999, which is a Continuation of U.S.application Ser. No. 08/463,261, filed Jun. 5, 1995 now U.S. Pat. No.6,448,642 which is a continuation-in-part of PCT/US94/14498, filed Dec.13, 1994 (filed in English), each of which are hereby incorporated byreference in their entireties. This application also claims benefitunder 35 U.S.C. § 119(e), of U.S. Provisional Application Nos.60/217,419, filed Jul. 11, 2000, and No. 60/220,829, filed Jul. 26,2000, each of which are hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides,polypeptides, and antibodies, as well as the production of suchpolynucleotides and polypeptides. More particularly, the polypeptides ofthe present invention are human tissue inhibitor of metalloproteinase-4polypeptides, hereinafter referred to as “human TIMP-4”. The inventionalso relates to inhibiting the action of such polypeptides.

BACKGROUND OF THE INVENTION

The extracellular matrix is a complex structure that contains collagen,proteoglycan, glycosaminoglycan, glycoproteins (fibronectin,chondronectin, laminin) and in some tissues, elastin (Hay, E. D., J.Cell Biol., 91:205-223 (1981)).

Matrix metalloproteinases (MMP's) constitute the major group ofzinc-binding endopeptidases that degrade extracellular matrix proteins,for example connective tissue, collagen and gelatin, during remodelingof connective tissue during normal physiological and some pathologicalprocesses. The unrestrained activity of MMP's may result in extensivetissue damage, and these enzymes have been implicated in a variety ofdisease processes, including tumor cell invasion, tumor angiogenesis andrheumatoid arthritis (Okada, Y., et al., J. Biol. Chem., 261:14245-14255(1986)). The MMP's are secreted from cells as inactive zymogens andtheir activity in the extracellular environment is regulated by variousactivators and inhibitors (Matrisian, L. M., Trends Genet., 6:121-125(1990)).

Regulation of metalloproteinase-mediated proteolysis may occur bynaturally occurring inhibitor proteins, such as tissue inhibitor ofmetalloproteinase (TIMP). The balance between the production andactivation of the MMP's, and their inhibition by natural inhibitors suchas TIMP, determines, in both physiological and pathological conditions,whether connective tissue is degraded.

MMP's include a number of proteases, exemplified by interstitial (typeI) collagenase itself, the stromelysins (also known as proteoglycanasesor transins), fibroblast and polymorphonuclear leukocyte gelatinases(also known as collagen-IV-ases), and pump-1 (putative metalloproteases1, uterine metalloproteases) [Goldberg et al, J. Biol. Chem. 2610:6600(1986); Whitham et al, Biochem. J. 240:913 (1986); Breathnach et al,Nucleic Acids Res., 15:1139 (1987); Muller et al, Biochem. J., 253:187(1988); Collier et al, J. Biol. Chem., 263:6579 (1988); Murphy et al,Biochem. J., 258:463 (1989); Quantin et al, Biochem. (N.Y.), 28:5327(1989); Birkedal-Hansen, J. Oral Pathol., 17:445 (1988)].

In general, the mammalian family of proteases has one or more of thefollowing properties: (a) optimal proteolytic activity around neutralpH; (b) dependence of the enzyme's activity on the presence of zinc, asevident by the loss of activity on treatment with divalent metal ionchelators, such as 1.10 phenanthroline (preferential chelation of zinc),or EDTA (less restricted chelating properties; EDTA and EGTA alsocontribute to enzyme inactivation via chelation of calcium ions requiredfor enzyme stability); (c) inhibition by TIMPs; (d) absence ofsignificant inhibition by known inhibitors of other families of neutral,zinc-containing metalloproteases, such as thermolysis,angiotensin-converting enzyme and ‘enkephalinases’; and (e) biosynthesisand secretion as latent precursor forms (zymogens), requiringextracellular activation. Activation has been achieved by a number ofendoproteases, organomercurials and chaotropic agents.

In general, members of the family of neutral metalloprotease enzymeshave distinctive substrate specificities. Thus, collagenase type I isunique in its ability to cleave a specific peptide bond within thenatural fibrils of the interstitial collagens (e.g. types I, II andIII). The gelatinases are only poorly active on these collagens, but areable to degrade denatured interstitial collagens, as well as thenon-fibrillar collagens, e.g. type IV, such as are found in the basementmembrane. Pump 1 has been reported to act preferentially on denaturedcollagens (gelatins), though its profile differs from that of thestromelysins or the collagenases type IV. Both the stromelysins and thegelatinases are also capable of degrading non-collagenous structuralproteins, such as the core protein of proteoglycan and elastin.Macromolecules involved in cell-to-substratum and cell-to-cellinteractions, such as laminin and fibronectin, are also susceptible todegradation by several of these metalloproteases.

Enzymes of this family are produced by synovial and skin fibroblasts,chondrocytes, peripheral mononuclear cells, keratinocytes and gingivaltissue, as well as existing within granule storage vesicles inpolymorphonuclear leukocytes (PMNLs).

Current information suggests that there is a family of metalloproteinaseinhibitors which comprises TIMP-1 (tissue inhibitor ofmetalloproteinases-1); TIMP-2; human TIMP-3 which has been cloned,expressed and mapped to human chromosome 22; and chicken tissueinhibitor of metalloproteinase (ChIMP-5). The polypeptide of the presentinvention has been putatively identified as a novel human TIMPpolypeptide based on amino acid sequence homology.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide which is human TIMP-4, as well asbiologically active and diagnostically or therapeutically usefulfragments, analogs and derivatives thereof.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding human TIMP-4,including mRNA's, DNA's, cDNA's, genomic DNA as well as biologicallyactive and diagnostically or therapeutically useful fragments, analogsand derivatives thereof.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptide by recombinanttechniques which comprises culturing recombinant prokaryotic and/oreukaryotic host cells, containing a human TIMP-4 nucleic acid sequenceunder conditions promoting expression of protein and subsequent recoveryof said protein.

In accordance with yet a further aspect of the present invention, thereis provided a method for treating conditions which are related toinsufficient human TIMP-4 activity which comprises administering to apatient in need thereof a pharmaceutical composition containing thehuman TIMP-4 protein of the invention which is effective to supplement apatient's endogenous human TIMP-4 and thereby alleviate said conditionswhich include, for example, arthritic diseases such as rheumatoid andosteoarthritis, soft tissue rheumatism, polychondritis and tendonitis;bone resorption diseases, such as osteoporosis, Paget's disease,hyperparathyroidism and cholesteatoma; the enhanced collagen destructionthat occurs in association with diabetes; the recessive classes ofdystrophic epidermolysis bullosa; periodontal disease, alveolitis andrelated consequences of gingival production of collagenase; cornealulceration; ulceration of the skin and gastrointestinal tract andabnormal wound healing; post-operative conditions in which collagenaselevels are raised; cancer by blocking the destruction of tissue basementmembranes leading to cancer metastasis; demyelinating diseases of thecentral and peripheral nervous systems; asthma; glomerulosclerosis;septic shock and infection; and psoriasis.

In accordance with yet a further aspect of the present invention, thereis provided a method for treating or preventing restenosis, whichcomprises administering to a patient in need thereof a pharmaceuticalcomposition containing the human TIMP-4 protein of the invention whichis effective to treat or prevent restenosis.

In accordance with yet a further aspect of the present invention, thereis provided an antibody against such polypeptides.

In accordance with yet another aspect of the present invention, thereare provided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to human TIMP-4 sequences.

In accordance with yet another aspect of the present invention, thereare provided antagonists to such polypeptides which may be employed fortherapeutic purposes, for example, for remodeling and repairing tissueand for destruction of scar tissue.

In accordance with another aspect of the present invention, there areprovided diagnostic assays for detecting diseases related to mutationsin human TIMP-4 sequences and over-expression of the polypeptide.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B shows the cDNA sequence and corresponding deduced amino acidsequence of the full-length human TIMP-4 polypeptide. The standardone-letter abbreviations for amino acids are used. Sequencing wasperformed using a 373 Automated DNA sequencer (Applied Biosystems,Inc.). Sequencing accuracy is predicted to be greater than 97% accurate.

FIGS. 2A-B is an amino acid sequence comparison between the polypeptideof the present invention and other human TIMP polypeptides.

FIGS. 3A-F shows the adenoviral plasmid maps used in the gene therapyexperiments described in Example 7.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with an aspect of the present invention, there is providedan isolated nucleic acid (polynucleotide) which encodes for the maturepolypeptide having the deduced amino acid sequence of FIGS. 1A-B or forthe mature polypeptide encoded by the cDNA of the clone deposited asATCC Deposit No. 75946 on Nov. 11, 1994.

A polynucleotide encoding a polypeptide of the present invention may beobtained from an early stage human brain. This contains an open readingframe and coding of protein of 224 amino acid residues of whichapproximately the first 29 residues represent the leader sequence suchthat the mature protein comprises 195 amino acid residues. Thepolynucleotide of this invention was discovered in a cDNA libraryderived from an early stage human brain. The protein exhibits thehighest degree of homology to Human TIMP-2 with 48% identity and 72%similarity over a 136 amino acid stretch. Human TIMP-4 has the signature12 cysteine amino acids, which are conserved in all members of the TIMPfamily. The 12 cysteine residues are all disulfide-linked in TIMP-1 andTIMP-2. This evidence strongly suggests that the polypeptide of thepresent invention is a novel member of the TIMP family.

The polynucleotide of the present invention may be in the form of RNA orin the form of DNA, which DNA includes cDNA, genomic DNA, and syntheticDNA. The DNA may be double-stranded or single-stranded, and if singlestranded may be the coding strand or non-coding (anti-sense) strand. Thecoding sequence which encodes the mature polypeptide may be identical tothe coding sequence shown in FIGS. 1A-B or that of the deposited cloneor may be a different coding sequence which coding sequence, as a resultof the redundancy or degeneracy of the genetic code, encodes the same,mature polypeptide as the DNA of FIGS. 1A-B or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of FIGS.1A-B or for the mature polypeptide encoded by the deposited cDNA mayinclude: only the coding sequence for the mature polypeptide; the codingsequence for the mature polypeptide and additional coding sequence suchas a leader or secretory sequence or a proprotein sequence; the codingsequence for the mature polypeptide (and optionally additional codingsequence) and non-coding sequence, such as introns or non-codingsequence 5′ and/or 3′ of the coding sequence for the mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIGS. 1A-B or the polypeptide encoded by the cDNA of the depositedclone. The variant of the polynucleotide may be a naturally occurringallelic variant of the polynucleotide or a non-naturally occurringvariant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIGS. 1A-B or the same mature polypeptideencoded by the cDNA of the deposited clone as well as variants of suchpolynucleotides which variants encode for a fragment, derivative oranalog of the polypeptide of FIGS. 1A-B or the polypeptide encoded bythe cDNA of the deposited clone. Such nucleotide variants includedeletion variants, substitution variants and addition or insertionvariants.

As hereinabove indicated, the polynucleotide may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIGS. 1A-B or of the coding sequence of the deposited clone. Asknown in the art, an allelic variant is an alternate form of apolynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptide may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains. Thus,for example, the polynucleotide of the present invention may encode fora mature protein, or for a protein having a prosequence or for a proteinhaving both a prosequence and a presequence (leader sequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

Fragments of the full length gene of the present invention may be usedas a hybridization probe for a cDNA library to isolate the full lengthcDNA and to isolate other cDNAs which have a high sequence similarity tothe gene or similar biological activity. Probes of this type preferablyhave at least 30 bases and may contain, for example, 50 or more bases.The probe may also be used to identify a cDNA clone corresponding to afull length transcript and a genomic clone or clones that contain thecomplete gene including regulatory and promotor regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, in oneembodiment, the term “stringent conditions” means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences. The polynucleotides which hybridize to thehereinabove described polynucleotides in a preferred embodiment encodepolypeptides which either retain substantially the same biologicalfunction or activity as the mature polypeptide encoded by the cDNAs ofFIGS. 1A-B (SEQ ID NO:1) or the deposited cDNA(s). In an alternativeembodiment, by “stringent hybridization conditions” is intendedovernight incubation at 42° C. in a solution comprising: 50% formamide,5× SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate(pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/mldenatured, sheared salmon sperm DNA, followed by washing the filters in0.1× SSC at about 65° C.

Alternatively, the polynucleotide may have at least 20 bases, preferably30 bases, and more preferably at least 50 bases which hybridize to apolynucleotide of the present invention and which has an identitythereto, as hereinabove described, and which may or may not retainactivity. For example, such polynucleotides may be employed as probesfor the polynucleotide of SEQ ID NO:1, for example, for recovery of thepolynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 80%, at least 85%, at least90%, and more preferably at least a 95% identity, at least a 96%identity, at least a 97% identity, at least a 98% identity, or at leasta 99% identity, to a polynucleotide which encodes the polypeptide of SEQID NO:2 as well as fragments thereof, which fragments have at least 30bases and preferably at least 50 bases and to polypeptides encoded bysuch polynucleotides. The deposit(s) referred to herein will bemaintained under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Micro-organisms for purposes of PatentProcedure. These deposits are provided merely as convenience to those ofskill in the art and are not an admission that a deposit is requiredunder 35 U.S.C. §112. The sequence of the polynucleotides contained inthe deposited materials, as well as the amino acid sequence of thepolypeptides encoded thereby, are incorporated herein by reference andare controlling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” or “identity” to a reference nucleotide sequenceencoding a TIMP-4 polypeptide is intended that the nucleotide sequenceof the polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five mismatches per each100 nucleotides of the reference nucleotide sequence encoding the TIMP-4polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. The reference (query) sequence may be the entirenucleotide sequence encoding TIMP-4, as shown in FIGS. 1A and 1B (SEQ IDNO:1) or any TIMP-4 polynucleotide sequence described herein.

As a practical matter, whether any particular nucleic acid molecule isat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the nucleotide sequences shown in FIGS. 1A and 1B, or to thecDNA sequence of the deposited cDNA clone, or fragments thereof, can bedetermined conventionally using known computer programs such as theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). Bestfit uses the local homology algorithmof Smith and Waterman to find the best segment of homology between twosequences (Advances in Applied Mathematics 2:482-489 (1981)). When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference nucleotide sequence and that gaps in homology ofup to 5% of the total number of nucleotides in the reference sequenceare allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag and colleagues(Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the queryand subject sequences are both DNA sequences. An RNA sequence can becompared by converting U's to T's. The result of said global sequencealignment is in percent identity. Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter. According to this embodiment, if thesubject sequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions, a manual correction ismade to the results to take into consideration the fact that the FASTDBprogram does not account for 5′ and 3′ truncations of the subjectsequence when calculating percent identity. For subject sequencestruncated at the 5′ or 3′ ends, relative to the query sequence, thepercent identity is corrected by calculating the number of bases of thequery sequence that are 5′ and 3′ of the subject sequence, which are notmatched/aligned, as a percent of the total bases of the query sequence.A determination of whether a nucleotide is matched/aligned is determinedby results of the FASTDB sequence alignment. This percentage is thensubtracted from the percent identity, calculated by the above FASTDBprogram using the specified parameters, to arrive at a final percentidentity score. This corrected score is what is used for the purposes ofthis embodiment. Only bases outside the 5′ and 3′ bases of the subjectsequence, as displayed by the FASTDB alignment, which are notmatched/aligned with the query sequence, are calculated for the purposesof manually adjusting the percent identity score. For example, a 90 basesubject sequence is aligned to a 100 base query sequence to determinepercent identity. The deletions occur at the 5′ end of the subjectsequence and therefore, the FASTDB alignment does not show amatched/alignment of the first 10 bases at 5′ end. The 10 unpaired basesrepresent 10% of the sequence (number of bases at the 5′ and 3′ ends notmatched/total number of bases in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 bases were perfectly matched the finalpercent identity would be 90%. In another example, a 90 base subjectsequence is compared with a 100 base query sequence. This time thedeletions are internal deletions so that there are no bases on the 5′ or3′ of the subject sequence which are not matched/aligned with the query.In this case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only bases 5′ and 3′ of the subject sequencewhich are not matched/aligned with the query sequence are manuallycorrected for. No other manual corrections are made for the purposes ofthis embodiment.

The present application is directed to nucleic acid molecules at least80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleicacid sequences (i.e., polynucleotides) disclosed herein (e.g., thosedisclosed in FIGS. 1A and 1B (SEQ ID NO:1) or to the cDNA sequence ofthe deposited clone), irrespective of whether they encode a polypeptidehaving TIMP-4 functional activity (e.g., biological activity). This isbecause even where a particular nucleic acid molecule does not encode apolypeptide having TIMP-4 activity, one of skill in the art would stillknow how to use the nucleic acid molecule, for instance, as ahybridization probe or a polymerase chain reaction (PCR) primer. Uses ofthe nucleic acid molecules of the present invention that do not encode apolypeptide having TIMP-4 activity include, inter alia, (1) isolatingthe TIMP-4 gene or allelic variants thereof in a cDNA library; (2) insitu hybridization (e.g., “FISH”) to metaphase chromosomal spreads toprovide precise chromosomal location of the TIMP-4 gene, as described inVerma et al., Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York (1988); and Northern Blot analysis for detecting TIMP-4mRNA expression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleicacid sequences disclosed herein (e.g., the nucleotide sequence shown inFIGS. 1A and 1B (SEQ ID NO:1) and the cDNA sequence of the depositedclone, or fragments thereof), which do, in fact, encode a polypeptidehaving TIMP-4 polypeptide functional activity (e.g., biologicalactivity).

By “a polypeptide having TIMP-4 functional activity” (e.g., biologicalactivity) is intended polypeptides exhibiting activity similar, but notnecessarily identical, to an activity of TIMP-4 polypeptides of theinvention, as measured in a particular functional assay. TIMP-4“functional activities include, but are not limited to, biologicalactivity (e.g., ability to inhibit metalloproteinase activity, abilityto inhibit the proliferation of cardiac smooth muscles, ability toinhibit the formation of the inner lining (neotima) of the carotidartery following balloon angioplasty injury), antigenicity [ability tobind (or compete with a TIMP-4 polypeptide for binding) to ananti-TIMP-4 antibody,], immunogenicity (ability to generate antibodywhich binds to a TIMP-4 polypeptide), and ability to bind to a TIMP-4receptor/ligand. Techniques known in the art may be applied to routinelydetermine if polypeptides of the invention exhibit TIMP-4 functionalactivities (e.g., biological activity (e.g., ability to inhibitmetalloproteinases (e.g., metalloproteinase 1, 2, 3, 7, and 9))).

In specific embodiments, the polynucleotides of the invention comprise,or alternatively consist of, a polynucleotide sequence that is at least10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, or 200, contiguous nucleotides of SEQ ID NO:1.Polypeptides encoded by these polynucleotides are also encompassed bythe invention.

In specific embodiments, the polynucleotides of the invention comprise,or alternatively consist of, a polynucleotides sequence encoding apolypeptide sequence that is at least 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200, contiguousamino acids of SEQ ID NO:2. Polypeptides encoded by thesepolynucleotides are also encompassed by the invention.

In specific embodiments, the polynucleotides of the invention comprise,or alternatively consist of, a nucleotide sequence encoding apolypeptide sequence selected from the group: (a) a polypeptide havingthe amino acid sequence of amino acids 22 to 28 of SEQ ID NO:2; and (b)a polypeptide having the amino acid sequence of amino acids 34 to 40 ofSEQ ID NO:2. Polypeptides encoded by these polynucleotides are alsoencompassed by the invention.

In specific embodiments, the polynucleotides of the invention comprise,or alternatively consist of, a nucleotide sequence encoding apolypeptide sequence selected from the group: (a) a polypeptide havingthe amino acid sequence of amino acids 1 to 72 of SEQ ID NO:2; (b) apolypeptide having the amino acid sequence of amino acids 73 to 127 ofSEQ ID NO:2; (c) a polypeptide having the amino acid sequence of aminoacids 128 to 176 of SEQ ID NO:2; and (d) a polypeptide having the aminoacid sequence of amino acids 1 to 176 of SEQ ID NO:2. Polypeptidesencoded by these polynucleotides are also encompassed by the invention.

In specific embodiments, the polynucleotides of the invention comprise,or alternatively consist of, a nucleotide sequence encoding apolypeptide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to a polypeptide sequence selected from thegroup: (a) a polypeptide having the amino acid sequence of amino acids 1to 72 of SEQ ID NO:2; (b) a polypeptide having the amino acid sequenceof amino acids 73 to 127 of SEQ ID NO:2; (c) a polypeptide having theamino acid sequence of amino acids 128 to 176 of SEQ ID NO:2; and (d) apolypeptide having the amino acid sequence of amino acids 1 to 176 ofSEQ ID NO:2. Polypeptides encoded by these polynucleotides are alsoencompassed by the invention.

The present invention further relates to a human TIMP-4 polypeptidewhich has the deduced amino acid sequence of FIGS. 1A-B or which has theamino acid sequence encoded by the deposited cDNA, as well as fragments,analogs and derivatives of such polypeptide.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptide of FIGS. 1A-B or that encoded by the deposited cDNA, means apolypeptide which retains essentially the same biological function oractivity as such polypeptide. Thus, an analog includes a proproteinwhich can be activated by cleavage of the proprotein portion to producean active mature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIGS. 1A-B orthat encoded by the deposited cDNA may be (i) one in which one or moreof the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

In specific embodiments, the polypeptides of the invention comprise, oralternatively consist of, an amino acid sequence that is at least 10,20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, or 200, contiguous amino acids of SEQ ID NO:2. Polynucleotidesencoding these polypeptides are also encompassed by the invention.

In specific embodiments, the polypeptides of the invention comprise, oralternatively consist of, a polypeptide selected from the group: (a) apolypeptide having the amino acid sequence of amino acids 1 to 72 of SEQID NO:2; (b) a polypeptide having the amino acid sequence of amino acids73 to 127 of SEQ ID NO:2; (c) a polypeptide having the amino acidsequence of amino acids 128 to 176 of SEQ ID NO:2; and (d) a polypeptidehaving the amino acid sequence of amino acids 1 to 176 of SEQ ID NO:2.Poynucleotides encoding these polypeptides are also encompassed by theinvention.

In specific embodiments, the polypeptides of the invention comprise, oralternatively consist of, a polypeptide selected from the group: (a) apolypeptide having the amino acid sequence of amino acids 22 to 28 ofSEQ ID NO:2; and (b) a polypeptide having the amino acid sequence ofamino acids 34 to 40 of SEQ ID NO:2. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

Preferred polypeptide fragments of the invention include the secretedprotein as well as the mature form. Further preferred polypeptidefragments include the secreted protein or the mature form having acontinuous series of deleted residues from the amino or the carboxyterminus, or both. Poynucleotides encoding these polypeptides are alsoencompassed by the invention.

Accordingly, polypeptide fragments include the secreted TIMP-4 proteinas well as the mature form. Further preferred polypeptide fragmentsinclude the secreted TIMP-4 protein or the mature form having acontinuous series of deleted residues from the amino or the carboxyterminus, or both. For example, any number of amino acids, ranging from1-29 of the TIMP-4 sequence disclosed in FIGS. 1A-B, can be deleted fromthe amino terminus of either the secreted TIMP-4 polypeptide or themature form. Similarly, any number of amino acids, ranging from 1-30 ofthe TIMP-4 sequence disclosed in FIGS. 1A-B, can be deleted from thecarboxy terminus of the secreted TIMP-4 protein or mature form.Furthermore, any combination of the above amino and carboxy terminusdeletions are preferred. Similarly, polynucleotides encoding thesepolypeptide fragments are also preferred. Particularly, N-terminaldeletions of the TIMP-4 polypeptide can be described by the generalformula m-224, where m is an integer from 2-218, where m corresponds tothe position of the amino acid residue identified in FIGS. 1A-B. More inparticular, the invention provides polypeptides comprising, oralternatively consisting of, an amino acid sequence selected from: P-2to P-224; G-3 to P-224; S-4 to P-224; P-5 to P-224; R-6 to P-224; P-7 toP-224; A-8 to P-224; P-9 to P-224; S-10 to P-224; W-11 to P-224; V-12 toP-224; L-13 to P-224; L-14 to P-224; L-15 to P-224; R-16 to P-224; L-17to P-224; L-18 to P-224; A-19 to P-224; L-20 to P-224; L-21 to P-224;R-22 to P-224; P-23 to P-224; P-24 to P-224; G-25 to P-224; L-26 toP-224; G-27 to P-224; E-28 to P-224; A-29 to P-224; C-30 to P-224; S-31to P-224; C-32 to P-224; A-33 to P-224; P-34 to P-224; A-35 to P-224;H-36 to P-224; P-37 to P-224; Q-38 to P-224; Q-39 to P-224; H-40 toP-224; I-41 to P-224; C-42 to P-224; H-43 to P-224; S-44 to P-224; A-45to P-224; L-46 to P-224; V-47 to P-224; I-48 to P-224; R-49 to P-224;A-50 to P-224; K-51 to P-224; I-52 to P-224; S-53 to P-224; S-54 toP-224; E-55 to P-224; K-56 to P-224; V-57 to P-224; V-58 to P-224; P-59to P-224; A-60 to P-224; S-61 to P-224; A-62 to P-224; D-63 to P-224;P-64 to P-224; A-65 to P-224; D-66 to P-224; T-67 to P-224; E-68 toP-224; K-69 to P-224; M-70 to P-224; L-71 to P-224; R-72 to P-224; Y-73to P-224; E-74 to P-224; I-75 to P-224; K-76 to P-224; Q-77 to P-224;I-78 to P-224; K-79 to P-224; M-80 to P-224; F-81 to P-224; K-82 toP-224; G-83 to P-224; F-84 to P-224; E-85 to P-224; K-86 to P-224; V-87to P-224; K-88 to P-224; D-89 to P-224; V-90 to P-224; Q-91 to P-224;Y-92 to P-224; I-93 to P-224; Y-94 to P-224; T-95 to P-224; P-96 toP-224; F-97 to P-224; D-98 to P-224; S-99 to P-224; S-100 to P-224;L-101 to P-224; C-102 to P-224; G-103 to P-224; V-104 to P-224; K-105 toP-224; L-106 to P-224; E-107 to P-224; A-108 to P-224; N-109 to P-224;S-110 to P-224; Q-111 to P-224; K-112 to P-224; Q-113 to P-224; Y-114 toP-224; L-115 to P-224; L-116 to P-224; T-117 to P-224; G-118 to P-224;Q-119 to P-224; V-120 to P-224; L-121 to P-224; S-122 to P-224; D-123 toP-224; G-124 to P-224; K-125 to P-224; V-126 to P-224; F-127 to P-224;I-128 to P-224; H-129 to P-224; L-130 to P-224; C-131 to P-224; N-132 toP-224; Y-133 to P-224; I-134 to P-224; E-135 to P-224; P-136 to P-224;W-137 to P-224; E-138 to P-224; D-139 to P-224; L-140 to P-224; S-141 toP-224; L-142 to P-224; V-143 to P-224; Q-144 to P-224; R-145 to P-224;E-146 to P-224; S-147 to P-224; L-148 to P-224; N-149 to P-224; H-150 toP-224; H-151 to P-224; Y-152 to P-224; H-153 to P-224; L-154 to P-224;N-155 to P-224; C-156 to P-224; G-157 to P-224; C-158 to P-224; Q-159 toP-224; I-160 to P-224; T-161 to P-224; T-162 to P-224; C-163 to P-224;Y-164 to P-224; T-165 to P-224; V-166 to P-224; P-167 to P-224; C-168 toP-224; T-169 to P-224; I-170 to P-224; S-171 to P-224; A-172 to P-224;P-173 to P-224; N-174 to P-224; E-175 to P-224; C-176 to P-224; L-177 toP-224; W-178 to P-224; T-179 to P-224; D-180 to P-224; W-181 to P-224;L-182 to P-224; L-183 to P-224; E-184 to P-224; R-185 to P-224; K-186 toP-224; L-187 to P-224; Y-188 to P-224; G-189 to P-224; Y-190 to P-224;Q-191 to P-224; A-192 to P-224; Q-193 to P-224; H-194 to P-224; Y-195 toP-224; V-196 to P-224; C-197 to P-224; M-198 to P-224; K-199 to P-224;H-200 to P-224; V-201 to P-224; D-202 to P-224; G-203 to P-224; T-204 toP-224; C-205 to P-224; S-206 to P-224; W-207 to P-224; Y-208 to P-224;R-209 to P-224; G-210 to P-224; H-211 to P-224; L-212 to P-224; P-213 toP-224; L-214 to P-224; R-215 to P-224; K-216 to P-224; E-217 to P-224;F-218 to P-224; and V-219 to P-224; of the amino acid sequence in FIGS.1A-B (the amino acid position in FIGS. 1A-B correspond to that of thesequence in SEQ ID NO:2 plus 29). The present application is alsodirected to polypeptides comprising, or alternatively, consisting of, anamino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%identical to a polypeptide described above. The present invention alsoencompasses the above polypeptide sequences fused to a heterologouspolypeptide sequence. Polynucleotides encoding these polypeptides arealso encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to bind TIMP-4 ligand) may stillbe retained. For example, the ability of the shortened TIMP-4 mutein toinduce and/or bind to antibodies which recognize the complete or matureforms of the polypeptide generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the C-terminus. Whether a particular polypeptide lackingC-terminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that an TIMP-4 muteinwith a large number of deleted C-terminal amino acid residues may retainsome biological or immunogenic activities. In fact, peptides composed ofas few as six TIMP-4 amino acid residues may often evoke an immuneresponse.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the TIMP-4 polypeptide shown in FIG. 1 (SEQ ID NO:2), asdescribed by the general formula 1-n, where n is an integer from 6-219,where n corresponds to the position of amino acid residue identified inFIGS. 1A-B. More in particular, the invention provides polypeptidescomprising, or alternatively consisting of, an amino acid sequenceselected from: M-1 to Q-223; M-1 to V-222; M-1 to I-221; M-1 to D-220;M-1 to V-219; M-1 to F-21; M-1 to E-217; M-1 to K-216; M-1 to R-215; M-1to L-214; M-1 to P-213; M-1 to L-212; M-1 to H-211; M-1 to G-210; M-1 toR-209; M-1 to Y-208; M-1 to W-207; M-1 to S-206; M-1 to C-205; M-1 toT-204; M-1 to G-203; M-1 to D-202; M-1 to V-201; M-1 to H-200; M-1 toK-199; M-1 to M-198; M-1 to C-197; M-1 to V-196; M-1 to Y-195; M-1 toH-194; M-1 to Q-193; M-1 to A-192; M-1 to Q-191; M-1 to Y-190; M-1 toG-189; M-1 to Y-188; M-1 to L-187; M-1 to K-186; M-1 to R-185; M-1 toE-184; M-1 to L-183; M-1 to L-182; M-1 to W-181; M-1 to D-180; M-1 toT-179; M-1 to W-178; M-1 to L-177; M-1 to C-176; M-1 to E-175; M-1 toN-174; M-1 to P-173; M-1 to A-172; M-1 to S-171; M-1 to I-170; M-1 toT-169; M-1 to C-168; M-1 to P-167; M-1 to V-166; M-1 to T-165; M-1 toY-164; M-1 to C-163; M-1 to T-162; M-1 to T-161; M-1 to I-160; M-1 toQ-159; M-1 to C-158; M-1 to G-157; M-1 to C-156; M-1 to N-155; M-1 toL-154; M-1 to H-153; M-1 to Y-152; M-1 to H-151; M-1 to H-150; M-1 toN-149; M-1 to L-148; M-1 to S-147; M-1 to E-146; M-1 to R-145; M-1 toQ-144; M-1 to V-143; M-1 to L-142; M-1 to S-141; M-1 to L-140; M-1 toD-139; M-1 to E-138; M-1 to W-137; M-1 to P-136; M-1 to E-135; M-1 toI-134; M-1 to Y-133; M-1 to N-132; M-1 to C-131; M-1 to L-130; M-1 toH-129; M-1 to I-128; M-1 to F-127; M-1 to V-126; M-1 to K-125; M-1 toG-124; M-1 to D-123; M-1 to S-122; M-1 to L-121; M-1 to V-120; M-1 toQ-119; M-1 to G-118; M-1 to T-117; M-1 to L-116; M-1 to L-115; M-1 toY-114; M-1 to Q-113; M-1 to K-112; M-1 to Q-111; M-1 to S-110; M-1 toN-109; M-1 to A-108; M-1 to E-107; M-1 to L-106; M-1 to K-105; M-1 toV-104; M-1 to G-103; M-1 to C-102; M-1 to L-101; M-1 to S-100; M-1 toS-99; M-1 to D-98; M-1 to F-97; M-1 to P-96; M-1 to T-95; M-1 to Y-94;M-1 to I-93; M-1 to Y-92; M-1 to Q-91; M-1 to V-90; M-1 to D-89; M-1 toK-88; M-1 to V-87; M-1 to K-86; M-1 to E-85; M-1 to F-84; M-1 to G-83;M-1 to K-82; M-1 to F-81; M-1 to M-80; M-1 to K-79; M-1 to I-78; M-1 toQ-77; M-1 to K-76; M-1 to I-75; M-1 to E-74; M-1 to Y-73; M-1 to R-72;M-1 to L-71; M-1 to M-70; M-1 to K-69; M-1 to E-68; M-1 to T-67; M-1 toD-66; M-1 to A-65; M-1 to P-64; M-1 to D-63; M-1 to A-62; M-1 to S-61;M-1 to A-60; M-1 to P-59; M-1 to V-58; M-1 to V-57; M-1 to K-56; M-1 toE-55; M-1 to S-54; M-1 to S-53; M-1 to I-52; M-1 to K-51; M-1 to A-50;M-1 to R-49; M-1 to I-48; M-1 to V-47; M-1 to L-46; M-1 to A-45; M-1 toS-44; M-1 to H-43; M-1 to C-42; M-1 to I-41; M-1 to H-40; M-1 to Q-39;M-1 to Q-38; M-1 to P-37; M-1 to H-36; M-1 to A-35; M-1 to P-34; M-1 toA-33; M-1 to C-32; M-1 to S-31; M-1 to C-30; M-1 to A-29; M-1 to E-28;M-1 to G-27; M-1 to L-26; M-1 to G-25; M-1 to P-24; M-1 to P-23; M-1 toR-22; M-1 to L-21; M-1 to L-20; M-1 to A-19; M-1 to L-18; M-1 to L-17;M-1 to R-16; M-1 to L-15; M-1 to L-14; M-1 to L-13; M-1 to V-12; M-1 toW-11; M-1 to S-10; M-1 to P-9; M-1 to A-8; and M-1 to P-7; of FIGS. 1A-B(the amino acid position in FIGS. 1A-B correspond to that of thesequence in SEQ ID NO:2 plus 29). The present application is alsodirected to polypeptides comprising, or alternatively, consisting of, anamino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, or 99%identical to a polypeptide described above. The present invention alsoencompasses the above polypeptide sequences fused to a heterologouspolypeptide sequence. Polynucleotides encoding these polypeptides arealso encompassed by the invention.

In further embodiments, the present invention encompasses polypeptidescomprising, or alternatively consisting of, an epitope of thepolypeptide having an amino acid sequence of SEQ ID NO:2, or an epitopeof the polypeptide sequence encoded by a polynucleotide sequencecontained in deposited clone 75946 or encoded by a polynucleotide thathybridizes to the complement of the sequence of SEQ ID NO:1 or containedin deposited clone 75946 under stringent hybridization conditions asdefined supra. The present invention further encompasses polynucleotidesequences encoding an epitope of a polypeptide sequence of the invention(such as, for example, the sequence disclosed in SEQ ID NO:1),polynucleotide sequences of the complementary strand of a polynucleotidesequence encoding an epitope of the invention, and polynucleotidesequences which hybridize to the complementary strand under stringenthybridization conditions or lower stringency hybridization conditionsdefined supra. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope,” as used herein, is defined as a portion of a protein thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed infra. (See, for example, Geysen et al., Proc. Natl. Acad.Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as usedherein, is defined as a portion of a protein to which an antibody canimmunospecifically bind its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Immunospecific binding excludes non-specific binding but does notnecessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

Fragments that function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 15, at least20, at least 25, and, most preferably, between about 15 to about 30amino acids. Preferred polypeptides comprising immunogenic or antigenicepitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Antigenicepitopes are useful, for example, to raise antibodies, includingmonoclonal antibodies, that specifically bind the epitope. Antigenicepitopes can be used as the target molecules in immunoassays. (See, forinstance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al.,Science 219:660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). A preferred immunogenic epitope includes thesecreted TIMP-4 protein. The polypeptides comprising one or moreimmunogenic epitopes may be presented for eliciting an antibody responsetogether with a carrier protein, such as an albumin, to an animal system(such as, for example, rabbit or mouse), or, if the polypeptide is ofsufficient length (at least about 25 amino acids), the polypeptide maybe presented without a carrier. However, immunogenic epitopes comprisingas few as 8 to 10 amino acids have been shown to be sufficient to raiseantibodies capable of binding to, at the very least, linear epitopes ina denatured polypeptide (e.g., in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as, for example, rabbits, rats, and miceare immunized with either free or carrier-coupled peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 micrograms of peptide or carrier protein andFreund's adjuvant or any other adjuvant known for stimulating an immuneresponse. Several booster injections may be needed, for instance, atintervals of about two weeks, to provide a useful titer of anti-peptideantibody that can be detected, for example, by ELISA assay using freepeptide adsorbed to a solid surface. The titer of anti-peptideantibodies in serum from an immunized animal may be increased byselection of anti-peptide antibodies, for instance, by adsorption to thepeptide on a solid support and elution of the selected antibodiesaccording to methods well known in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to other polypeptide sequences. Forexample, the polypeptides of the present invention may be fused with theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof)resulting in chimeric polypeptides. Such fusion proteins may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature, 331:84-86 (1988). IgG Fusion proteins thathave a disulfide-linked dimeric structure due to the IgG portiondesulfide bonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix-binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni²⁺nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, alteration of polynucleotides corresponding to SEQ IDNO:1 and the polypeptides encoded by these polynucleotides may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments by homologous or site-specific recombination togenerate variation in the polynucleotide sequence. In anotherembodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide coding apolypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

The polypeptides of the present invention include the polypeptide of SEQID NO:2 (in particular the mature polypeptide) as well as polypeptideswhich have at least 70% similarity (preferably at least 70% identity) tothe polypeptide of SEQ ID NO:2 and more preferably at least 90%similarity (more preferably at least 80% identity, or at least 85%identity) to the polypeptide of SEQ ID NO:2 and still more preferably atleast 95% similarity (still more preferably at least 90% identity, atleast 95% identity, at least 96% identity, at least 97% identity, atleast 98% identity, or at least 99% identity ) to the polypeptide of SEQID NO:2 and also include portions of such polypeptides with such portionof the polypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” or “identity” to a reference amino acid sequence of aTIMP-4 polypeptide is intended that the amino acid sequence of thepolypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of the TIMP-4polypeptide. In other words, to obtain a polypeptide having an aminoacid sequence at least 95% identical to a reference amino acid sequence,up to 5% of the amino acid residues in the reference sequence may bedeleted or substituted with another amino acid, or a number of aminoacids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, theamino acid sequence shown in FIGS. 1A and 1B (SEQ ID NO:2), the aminoacid sequence encoded by the deposited cDNA clone, or fragments thereof,can be determined conventionally using known computer programs such theBestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the reference aminoacid sequence and that gaps in homology of up to 5% of the total numberof amino acid residues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query)sequence (a sequence of the present invention) and a subject sequence,also referred to as a global sequence alignment, is determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDBamino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1,Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter. According to this embodiment, if the subject sequence isshorter than the query sequence due to N- or C-terminal deletions, notbecause of internal deletions, a manual correction is made to theresults to take into consideration the fact that the FASTDB program doesnot account for N- and C-terminal truncations of the subject sequencewhen calculating global percent identity. For subject sequencestruncated at the N- and C-termini, relative to the query sequence, thepercent identity is corrected by calculating the number of residues ofthe query sequence that are N- and C-terminal of the subject sequence,which are not matched/aligned with a corresponding subject residue, as apercent of the total bases of the query sequence. A determination ofwhether a residue is matched/aligned is determined by results of theFASTDB sequence alignment. This percentage is then subtracted from thepercent identity, calculated by the above FASTDB program using thespecified parameters, to arrive at a final percent identity score. Thisfinal percent identity score is what is used for the purposes of thisembodiment. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence. For example, a 90 aminoacid residue subject sequence is aligned with a 100 residue querysequence to determine percent identity. The deletion occurs at theN-terminus of the subject sequence and therefore, the FASTDB alignmentdoes not show a matching/alignment of the first 10 residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N- and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.In another example, a 90 residue subject sequence is compared with a 100residue query sequence. This time the deletions are internal deletionsso there are no residues at the N- or C-termini of the subject sequencewhich are not matched/aligned with the query. In this case the percentidentity calculated by FASTDB is not manually corrected. Once again,only residue positions outside the N- and C-terminal ends of the subjectsequence, as displayed in the FASTDB alignment, which are notmatched/aligned with the query sequence are manually corrected for. Noother manual corrections are made for the purposes of this embodiment.

In specific embodiments, the polypeptides of the invention comprise, oralternatively consist of, a polypeptitde that is at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% identical to a polypeptide selectedfrom the group: (a) a polypeptide having the amino acid sequence ofamino acids 1 to 72 of SEQ ID NO:2; (b) a polypeptide having the aminoacid sequence of amino acids 73 to 127 of SEQ ID NO:2; (c) a polypeptidehaving the amino acid sequence of amino acids 128 to 176 of SEQ ID NO:2;and (d) a polypeptide having the amino acid sequence of amino acids 1 to176 of SEQ ID NO:2. Poynucleotides encoding these polypeptides are alsoencompassed by the invention.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention. Thepresent invention also relates to vectors which include polynucleotidesof the present invention, host cells which are genetically engineeredwith vectors of the invention and the production of polypeptides of theinvention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the human TIMP-4 genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda PL promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 and Sf9;animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plantcells, etc. The selection of an appropriate host is deemed to be withinthe scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are PKK232-8 and PCM7. Particular namedbacterial promoters include lac, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation. (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), thedisclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), alpha-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The human TIMP-4 polypeptides can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (BPLC) can be employed for finalpurification steps.

The polypeptides of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

Antibodies

The present invention further relates to antibodies and T-cell antigenreceptors (TCR) which immunospecifically bind a polypeptide, preferablyan epitope, of the present invention (as determined by immunoassays wellknown in the art for assaying specific antibody-antigen binding).Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. The term“antibody,” as used herein, refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. The immunoglobulin molecules of the invention can beof any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.In specific embodiments, the immunoglobulin molecule is IgG1. In otherspecific embodiments, the immunoglobulin molecule is IgG4.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine, donkey, shiprabbit, goat, guinea pig, camel, horse, or chicken. As used herein,“human” antibodies include antibodies having the amino acid sequence ofa human immunoglobulin and include antibodies isolated from humanimmunoglobulin libraries or from animals transgenic for one or morehuman immunoglobulin and that do not express endogenous immunoglobulins,as described infra and, for example in, U.S. Pat. No. 5,939,598 byKucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention that they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies thatspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. Antibodies that do not bind polypeptides with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a polypeptide of the present invention are also included inthe present invention. Further included in the present invention areantibodies that bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻²M, 10⁻²M, 5×10⁻³M, 10⁻³M, 5×10⁻⁴M, 10⁻⁴M, 5×10⁻⁵M, 10⁻⁵M, 5×10⁻⁶M,10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M,5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M5×10⁻¹⁵M, and 10⁻¹⁵M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. In specific embodiments, the antagonistic antibodies of theinvention increase or enhance metalloproteinase activity.). In specificembodiments, antibodies are provided that increase or enhancemetalloproteinase activity by at least 90%, at least 80%, at least 70%,at least 60%, or at least 50% of the activity in absence of theantibody. In an alternative example, the present invention includesantibodies which enhance receptor/ligand interactions with thepolypeptides of the invention either partially or fully. In specificembodiments, the agonsitic antibodies of the invention decrease orreduce metalloproteinase activity. In specific embodiments, antibodiesare provided that decrease or reduce metalloproteinase activity by atleast 90%, at least 80%, at least 70%, at least 60%, or at least 50% ofthe activity in absence of the antibody. The invention features bothreceptor-specific antibodies and ligand-specific antibodies. Theinvention also features receptor-specific antibodies which do notprevent ligand binding but prevent receptor activation. Receptoractivation (i.e., signaling) may be determined by techniques describedherein or otherwise known in the art. For example, receptor activationcan be determined by detecting the phosphorylation (e.g., tyrosine orserine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra). In specific embodiments, antibodies are provided thatenhance or increase ligand or receptor activity by at least 90%, atleast 80%, at least 70%, at least 60%, or at least 50% of the activityin absence of the antibody. In specific embodiments, antibodies areprovided that inhibit ligand or receptor activity by at least 90%, atleast 80%, at least 70%, at least 60%, or at least 50% of the activityin absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation. The antibodiesmay be specified as agonists, antagonists or inverse agonists forbiological activities comprising the specific biological activities ofthe peptides of the invention disclosed herein. The above antibodyagonists can be made using methods known in the art. See, e.g., PCTpublication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood92(6):1981-1988 (1998); Chen, et al., Cancer Res. 58(16):3668-3678(1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al.,Cancer Res. 58(15):3209-3214 (1998); Yoon, et al., J. Immunol.160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 11(Pt2):237-247(1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997);Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol.Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762(1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al.,Cytokine 8(1):14-20 (1996) (which are all incorporated by referenceherein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, or toxins. See, e.g., PCT publicationsWO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP396,387.

The antibodies of the invention include derivatives that are modified,i.e, by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interestcan be produced by various procedures well known in the art. Forexample, a polypeptide of the invention can be administered to varioushost animals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well-known in the art and arediscussed in detail in Example 6. Briefly, mice can be immunized with apolypeptide of the invention or a cell expressing such peptide. Once animmune response is detected, e.g., antibodies specific for the antigenare detected in the mouse serum, the mouse spleen is harvested andsplenocytes isolated. The splenocytes are then fused by well-knowntechniques to any suitable myeloma cells, for example cells from cellline SP20 available from the ATCC. Hybridomas are selected and cloned bylimited dilution. The hybridoma clones are then assayed by methods knownin the art for cells that secrete antibodies capable of binding apolypeptide of the invention. Ascites fluid, which generally containshigh levels of antibodies, can be generated by immunizing mice withpositive hybridoma clones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular, such phage can be utilized to displayantigen-binding domains expressed from a repertoire or combinatorialantibody library (e.g., human or murine). Phage expressing an antigenbinding domain that binds the antigen of interest can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Phage used in these methods aretypically filamentous phage including fd and M13 binding domainsexpressed from phage with Fab, Fv or disulfide stabilized Fv antibodydomains recombinantly fused to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporatedherein by reference in their entireties. Humanized antibodies areantibody molecules from non-human species antibody that binds thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and framework regions from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat.Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; and 5,939,598, which are incorporated by reference herein intheir entirety. In addition, companies such as Abgenix, Inc. (Freemont,Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:2.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligation of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be obtained from a suitable source (e.g., an antibodycDNA library, or a cDNA library generated from, or nucleic acid,preferably poly A+ RNA, isolated from, any tissue or cells expressingthe antibody, such as hybridoma cells selected to express an antibody ofthe invention) by PCR amplification using synthetic primers hybridizableto the 3′ and 5′ ends of the sequence or by cloning using anoligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties ), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855;Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature314:452-454) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-54) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,1988, Science 242:1038-1041).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, e.g., a heavy or light chain of anantibody of the invention, requires construction of an expression vectorcontaining a polynucleotide that encodes the antibody. Once apolynucleotide encoding an antibody molecule or a heavy or light chainof an antibody, or portion thereof (preferably containing the heavy orlight chain variable domain), of the invention has been obtained, thevector for the production of the antibody molecule may be produced byrecombinant DNA technology using techniques well known in the art. Thus,methods for preparing a protein by expressing a polynucleotidecontaining an antibody encoding nucleotide sequence are describedherein. Methods which are well known to those skilled in the art can beused to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination. Theinvention, thus, provides replicable vectors comprising a nucleotidesequence encoding an antibody molecule of the invention, or a heavy orlight chain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, operably linked to aheterologous promoter. In preferred embodiments for the expression ofdouble-chained antibodies, vectors encoding both the heavy and lightchains may be co-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, NSO, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., 1986, Gene 45:101; Cockett et al., 1990,Bio/Technology 8:2).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.2:1791), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiationsignals may also be required for efficient translation of insertedantibody coding sequences. These signals include the ATG initiationcodon and adjacent sequences. Furthermore, the initiation codon must bein phase with the reading frame of the desired coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bittner et al., 1987, Methods inEnzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthineguanine phosphoribosyltransferase (Szybalska &Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan,1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215); and hygro,which confers resistance to hygromycin (Santerre et al., 1984, Gene30:147). Methods commonly known in the art of recombinant DNA technologywhich can be used are described in Ausubel et al. (eds.), 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, CurrentProtocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin etal., 1981, J. Mol. Biol. 150:1, which are incorporated by referenceherein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol.3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol.3:257).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain should be placedbefore the heavy chain to avoid an excess of toxic free heavy chain(Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci.USA 77:2197). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

Antibody Conjugates

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20 or 50 amino acids of the polypeptide) of the present invention togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences. The antibodies may bespecific for antigens other than polypeptides (or portion thereof,preferably at least 10, 20 or 50 amino acids of the polypeptide) of thepresent invention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etat., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

As discussed, supra, the polypeptides of the present invention may befused or conjugated to the above antibody portions to increase the invivo half life of the polypeptides or for use in immunoassays usingmethods known in the art. Further, the polypeptides of the presentinvention may be fused or conjugated to the above antibody portions tofacilitate purification. One reported example describes chimericproteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. (EP 394,827; Traunecker etal., Nature 331:84-86 (1988). The polypeptides of the present inventionfused or conjugated to an antibody having disulfide-linked dimericstructures (due to the IgG) may also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)). In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP A 232,262). Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995)0.

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitates theirpurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. See,for example, U.S. Pat. No. 4,741,900 for metal ions which can beconjugated to antibodies for use as diagnostics according to the presentinvention. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion. A cytotoxin orcytotoxic agent includes any agent that is detrimental to cells.Examples include paclitaxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 125I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest is conjugated to a labeled compound (e.g., 3H or125I) in the presence of increasing amounts of an unlabeled secondantibody.

Therapeutic Uses

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the described disorders. Therapeutic compounds of theinvention include, but are not limited to, antibodies of the invention(including fragments, analogs and derivatives thereof as describedherein) and nucleic acids encoding antibodies of the invention(including fragments, analogs and derivatives thereof as describedherein). The antibodies of the invention can be used to treat, inhibitor prevent diseases and disorders associated with aberrant expressionand/or activity of a polypeptide of the invention. The treatment and/orprevention of diseases and disorders associated with aberrant expressionand/or activity of a polypeptide of the invention includes, but is notlimited to, alleviating symptoms associated with those diseases anddisorders. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides,including fragments thereof. Preferred binding affinities include thosewith a dissociation constant or Kd less than 5×10−6 M, 10−6 M, 5×10−7 M,10−7 M, 5×10−8 M, 10−8 M, 5×10−9 M, 10−9 M, 5×10−10 M, 10−10 M, 5×10−11M, 10−11 M, 5×10−12 M, 10−12 M, 5×10−13 M, 10−13 M, 5×10−14 M, 10−14 M,5×10−15 M, and 10−15 M.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann.Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155-215). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), 1993, Current Protocols inMolecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY.

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody nucleic acids(Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935;Zijlstra et al., 1989, Nature 342:435-438). In specific embodiments, theexpressed antibody molecule is a single chain antibody; alternatively,the nucleic acid sequences include sequences encoding both the heavy andlight chains, or fragments thereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432)(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180 dated Apr. 16, 1992(Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilson et al.); WO92/20316dated Nov. 26, 1992 (Findeis et al.); WO93/14188 dated Jul. 22, 1993(Clarke et al.), WO 93/20221 dated Oct. 14, 1993 (Young)).Alternatively, the nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression, by homologousrecombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., 1993, Meth. Enzymol.217:581-599). These retroviral vectors have been to delete retroviralsequences that are not necessary for packaging of the viral genome andintegration into host cell DNA. The nucleic acid sequences encoding theantibody to be used in gene therapy are cloned into one or more vectors,which facilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., 1994, Biotherapy6:291-302, which describes the use of a retroviral vector to deliver themdr1 gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., 1994, J. Clin.Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons andGunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson,1993, Curr. Opin. in Genetics and Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3:499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5:3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT PublicationWO94/12649; and Wang, et al., 1995, Gene Therapy 2:775-783.

In cases where an adenovirus is used as an expression vector, theantibody coding sequence of interest may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingthe TIMP-4 molecule in infected hosts. (e.g., see Logan & Shenk, 1984,Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals mayalso be required for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see Bittner et al., 1987, Methods in Enzymol.153:51-544).

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300;U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth.Enzymol. 217:599-618; Cohen et al., 1993, Meth. Enzymol. 217:618-644;Cline, 1985, Pharmac. Ther. 29:69-92) and may be used in accordance withthe present invention, provided that the necessary developmental andphysiological functions of the recipient cells are not disrupted. Thetechnique should provide for the stable transfer of the nucleic acid tothe cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598, dated Apr. 28,1994; Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald, 1980,Meth. Cell Bio. 21A:229; and Pittelkow and Scott, 1986, Mayo ClinicProc. 61:771).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Composition

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part ofa retroviral or other vector, etc. Methods of introduction include butare not limited to intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, and oral routes. Thecompounds or compositions may be administered by any convenient route,for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local. Inaddition, it may be desirable to introduce the pharmaceutical compoundsor compositions of the invention into the central nervous system by anysuitable route, including intraventricular and intrathecal injection;intraventricular injection may be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir. Pulmonary administration can also be employed, e.g., by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, 1990, Science249:1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York,pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201;Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J.Med. 321:574). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol.Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al.,1989, Ann. Neurol. 25:351; Howard et al., 1989, J.Neurosurg. 71:105). Inyet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(1990, Science 249:1527-1533).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox—like peptide which is knownto enter the nucleus (see e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases and/ordisorders associated with the aberrant expression and/or activity of apolypeptide of the invention. The invention provides for the detectionof aberrant expression of a polypeptide of interest, comprising (a)assaying the expression of the polypeptide of interest in cells or bodyfluid of an individual using one or more antibodies specific to thepolypeptide interest and (b) comparing the level of gene expression witha standard gene expression level, whereby an increase or decrease in theassayed polypeptide gene expression level compared to the standardexpression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosising a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, M., et al., J. Cell.Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol.105:3087-3096 (1987)). Other antibody-based methods useful for detectingprotein gene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitableantibody assay labels are known in the art and include enzyme labels,such as, glucose oxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I),carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium(⁹⁹Tc); luminescent labels, such as luminol; and fluorescent labels,such as fluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide of theinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or calorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Therapeutic and Diagnostic Uses

The present invention is also directed, in part, to human TIMP-4 whichhas, as a defining characteristic, the ability to inhibit the action ofMMP's. The human TIMP-4 polypeptide may be employed as ametalloproteinase inhibitor to prevent tumor invasion and angiogenesesand subsequent metastases. The human TIMP-4 polypeptide may also beemployed to treat arthritic diseases, such as rheumatoid arthritis andosteoarthritis, soft tissue rheumatism, polychondritis and tendonitis;and bone resorption diseases, such as osteoporosis, Paget's disease,hyperparathyroidism and cholesteatoma. Human TIMP-4 may also be employedto prevent enhanced collagen destruction that occurs in association withdiabetes, the recessive classes of dystrophic epidermolysis bullosa,periodontal disease and related consequences of gingival production ofcollagenase. human TIMP-4 may also be employed to inhibit PMNLcollagenase release following cellular infiltration to inflamed gingiva,including combatting the greater susceptibility of diabetes patients toperiodontal disease.

Human TIMP-4 may also be employed to treat corneal ulceration, forexample, that induced by alkali or other burns, by radiation, by VitaminE or retinoid deficiency; ulceration of the skin and gastrointestinaltract, and abnormal wound healing, and post-operative conditionsincluding colonic anastomosis, in which collagenase levels are raised.

MMP's mediate tumor growth in situ. Accordingly, human TIMP-4 may beused to block the destruction of cellular basement membranes, which isthe mechanism by which cancer cells metastasize. MMP's have beenimplicated in neovascularization required to support tumor growth andsurvival, in the tissue remodeling required to accommodate the growingprimary and secondary tumors, and in the penetration of tumor cellsthrough the basement membranes of the vascular walls during metastasis.

Thus, in specific embodiments, the invention provides for treatment orprevention of various diseases and disorders involving cellproliferation, tumor cell invasion, and tumor angiogenesis.

TIMP-4 polynucleotides, polypeptides, antibodies (e.g., agonisticanti-TIMP-4 antibodies) and/or agonists of the invention, may be usedfor therapeutic/prophylactic purposes alone or in combination with othertherapeutics useful in the treatment of cancer and hyperproliferative ordysproliferative disorders. Diseases and disorders involving celloverproliferation are treated or prevented by administration of a TIMP-4polynucleotide, polypeptide, antibody and/or agonist of the inventionthat promotes (i.e., increases or supplies) TIMP-4 function.

Diseases and disorders involving cell overproliferation that can betreated or prevented using the polynucleotides, polypeptides,antibodies, and/or agonists of the invention include, but are notlimited to, malignancies, premalignant conditions (e.g., hyperplasia,metaplasia, dysplasia), benign tumors, hyperproliferative disorders,benign dysproliferative disorders, etc.

Malignancies and related disorders that can be treated or prevented byadministration of a TIMP-4 polynucleotide, polypeptide, antibody and/oragonist of the invention, include but are not limited to, leukemia,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia,myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia,monocytic leukemia, erythroleukemia, chronic leukemia, chronicmyelocytic (granulocytic) leukemia, chronic lymphocytic leukemia,Polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's disease,multiple myeloma, Waldenstrom's macroglobulinemia, Heavy chain disease,solid tumors, sarcomas and carcinomas, fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,cervical cancer, uterine cancer, testicular tumor, lung carcinoma, smallcell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, retinoblastoma, nasopharyngeal carcinoma, andesophageal carcinoma.(for a review of such disorders, see Fishman etal., 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia).

In a specific embodiment, digestive tract tumors are treated orprevented, including but not limited to, esophageal, stomach, colon, andcolorectal cancers. In another specific embodiment, airway cancers suchas lung cancers (e.g., small cell lung carcinoma) and nasopharyngealcarcinoma are treated or prevented. In yet other specific embodiments,malignancy or dysproliferative changes (such as metaplasias anddysplasias), or hyperproliferative disorders, are treated or preventedin the head, neck, cervix, kidney, stomach, skin, ovary, bladder,breast, colon, lung, or uterus. In other specific embodiments, sarcoma,or leukemia is treated or prevented. In another particular embodiment,osteosarcoma or renal cell carcinoma is treated or prevented.

TIMP-4 polynucleotides, polypeptides, antibodies and/or agonists of theinvention can also be administered to treat premalignant conditions andto prevent progression to a neoplastic or malignant state, including butnot limited to, those disorders listed above. Such prophylactic ortherapeutic use is indicated in conditions known or suspected ofpreceding progression to neoplasia or cancer, in particular, wherenon-neoplastic cell growth consisting of hyperplasia, metaplasia, ormost particularly, dysplasia has occurred (for review of such abnormalgrowth conditions, see Robbins and Angell, 1976, Basic Pathology, 2dEd., W. B. Saunders Co., Philadelphia, pp. 68-79.) Hyperplasia is a formof controlled cell proliferation involving an increase in cell number ina tissue or organ, without significant alteration in structure orfunction. As but one example, endometrial hyperplasia often precedesendometrial cancer. Metaplasia is a form of controlled cell growth inwhich one type of adult or fully differentiated cell substitutes foranother type of adult cell. Metaplasia can occur in epithelial orconnective tissue cells. Atypical metaplasia involves a somewhatdisorderly metaplastic epithelium. Dysplasia is frequently a forerunnerof cancer, and is found mainly in the epithelia; it is the mostdisorderly form of non-neoplastic cell growth, involving a loss inindividual cell uniformity and in the architectural orientation ofcells. Dysplastic cells often have abnormally large, deeply stainednuclei, and exhibit pleomorphism. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation, and is oftenfound in the cervix, respiratory passages, oral cavity, and gallbladder.

In a specific embodiment, a TIMP-4 polypeptide, polynucleotide,antibody, and/or agonist of the invention is administered to a humanpatient to prevent progression to breast, colon, lung, stomach oruterine cancer, or melanoma or sarcoma.

In another embodiment of the invention, a polynucleotide, polypeptide,antibody and/or agonist of the invention is used to treat or preventhyperproliferative or benign dysproliferative disorders. Specificembodiments are directed to treatment or prevention of benign tumors,fibrocystic conditions, and tissue hypertrophy (e.g., prostatichyperplasia).

In a specific embodiment TIMP-4 polynucleotides, polypeptides,antibodies, and/or agonists of the invention are administered to treator prevent an immune system related disease, disorder or condition. In apreferred embodiment, the immune system disease, disorder, or conditionis an autoimmune diesease, disorder, or condition. In a most preferredembodiment, the immune system diesease, disorder, or condition, isrheumatoid arthritis.

MMP's are responsible for localized degradation of the follicular wallduring ovulation and localized degradation of the uterine wall forblastocyte implantation. Accordingly, human TIMP-4 may be employed as acontraceptive.

Human TIMP-4 may also be employed as a general growth factor to treatrestenosis and similar diseases. Human TIMP-4 may be employedparticularly as a growth factor for erythroid cell lineages.

TIMP-4 is a strong inhibitor of metalloproteinases which degradestructural components of tissues and are involved in the remodeling oftissues in normal and certain pathological states. Accordingly,potential therapeutic applications of the TIMP-4 polynucleotides,polypeptides, antibodies, and/or agonists of the invention include, butare not limited to, the treatment and/or prevention of restenosis, orobstruction of blood vessels, such as coronary arteries, and heartfailure. Restenosis is a medical condition characterized by theconstriction of coronary arteries. Restenosis usually occurs followingtreatment to open coronary arteries clogged by plaque accumulation.Balloon angioplasty, insertion of a catheter into the clogged arteryfollowed by expansion of a balloon at the site of blockage, compressesthe plaque and opens the arteries. This procedure can damage the wall ofthe artery. The damaged vessel often responds to the balloon angioplastyinjury by overgrowth, which can lead to reconstriction of the artery.While not intending to be bound by theory, it is believed that TIMP-4acts to treat or prevent restenosis by blocking metalloproteinases, afamily of genes that become active after injury to the artery and arethought to play a major role in restenosis.

TIMP-4 polynucleotides or polypeptides, or agonists or antagonists ofTIMP-4, encoding TIMP-4 may be used to treat, prevent, and/or diagnosecardiovascular diseases, disorders, and/or conditions, includingperipheral artery disease, such as limb ischemia.

Cardiovascular diseases, disorders, and/or conditions that may betreated, prevented and/or diagnosed with the polynucleotides,polypeptides (including antibodies), agonists and/or antagonists of theinvention include, but are not limited to, cardiovascular abnormalities,such as arterio-arterial fistula, arteriovenous fistula, cerebralarteriovenous malformations, congenital heart defects, pulmonaryatresia, and Scimitar Syndrome. Congenital heart defects include aorticcoarctation, cor triatriatum, coronary vessel anomalies, crisscrossheart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly,Eisenmenger complex, hypoplastic left heart syndrome, levocardia,tetralogy of fallot, transposition of great vessels, double outlet rightventricle, tricuspid atresia, persistent truncus arteriosus, and heartseptal defects, such as aortopulmonary septal defect, endocardialcushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricularheart septal defects.

Cardiovascular diseases, disorders, and/or conditions that may betreated, prevented and/or diagnosed with the polynucleotides,polypeptides (including antibodies), agonists and/or antagonists of theinvention also include, but are not limited to, heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias that may be treated, prevented and/or diagnosed with thepolynucleotides, polypeptides (including antibodies), agonists and/orantagonists of the invention include, but are not lmited to, sinusarrhythmia, atrial fibrillation, atrial flutter, bradycardia,extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrialblock, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome,Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome,sick sinus syndrome, tachycardias, and ventricular fibrillation.Tachycardias include paroxysmal tachycardia, supraventriculartachycardia, accelerated idioventricular rhythm, atrioventricular nodalreentry tachycardia, ectopic atrial tachycardia, ectopic junctionaltachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia,Torsades de Pointes, and ventricular tachycardia.

Heart valve disease that may be treated, prevented and/or diagnosed withthe polynucleotides, polypeptides (including antibodies), agonistsand/or antagonists of the invention include, but are not limited to,aortic valve insufficiency, aortic valve stenosis, hear murmurs, aorticvalve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitralvalve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonaryvalve insufficiency, pulmonary valve stenosis, tricuspid atresia,tricuspid valve insufficiency, and tricuspid valve stenosis.

Myocardial diseases that may be treated, prevented and/or diagnosed withthe polynucleotides, polypeptides (including antibodies), agonistsand/or antagonists of the invention include, but are not limited to,alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophiccardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvularstenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardialfibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardialreperfusion injury, and myocarditis.

Myocardial ischemias that may be treated, prevented and/or diagnosedwith the polynucleotides, polypeptides (including antibodies), agonistsand/or antagonists of the invention include, but are not limited to,coronary disease, such as angina pectoris, coronary aneurysm, coronaryarteriosclerosis, coronary thrombosis, coronary vasospasm, myocardialinfarction and myocardial stunning.

Cardiovascular diseases that may be treated, prevented and/or diagnosedwith the polynucleotides, polypeptides (including antibodies), agonistsand/or antagonists of the invention also include, but are not limitedto, vascular diseases such as aneurysms, angiodysplasia, angiomatosis,bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-WeberSyndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases,Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusivediseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovasculardiseases, disorders, and/or conditions, diabetic angiopathies, diabeticretinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids,hepatic veno-occlusive disease, hypertension, hypotension, ischemia,peripheral vascular diseases, phlebitis, pulmonary veno-occlusivedisease, Raynaud's disease, CREST syndrome, retinal vein occlusion,Scimitar syndrome, superior vena cava syndrome, telangiectasia, ataciatelangiectasia, hereditary hemorrhagic telangiectasia, varicocele,varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

Aneurysms that may be treated, prevented and/or diagnosed with thepolynucleotides, polypeptides (including antibodies), agonists and/orantagonists of the invention include, but are not limited to, dissectinganeurysms, false aneurysms, infected aneurysms, ruptured aneurysms,aortic aneurysms, cerebral aneurysms, coronary aneurysms, heartaneurysms, and iliac aneurysms.

Arterial occlusive diseases that may be treated, prevented and/ordiagnosed with the polynucleotides, polypeptides (including antibodies),agonists and/or antagonists of the invention include, but are notlimited to, arteriosclerosis, intermittent claudication, carotidstenosis, fibromuscular dysplasias, mesenteric vascular occlusion,Moyamoya disease, renal artery obstruction, retinal artery occlusion,and thromboangiitis obliterans.

Cerebrovascular diseases, disorders, and/or conditions that may betreated, prevented and/or diagnosed with the polynucleotides,polypeptides (including antibodies), agonists and/or antagonists of theinvention include, but are not limited to, carotid artery diseases,cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebralartery diseases, cerebral embolism and thrombosis, carotid arterythrombosis, sinus thrombosis, Wallenberg's syndrome, cerebralhemorrhage, epidural hematoma, subdural hematoma, subaraxhnoidhemorrhage, cerebral infarction, cerebral ischemia (includingtransient), subclavian steal syndrome, periventricular leukomalacia,vascular headache, cluster headache, migraine, and vertebrobasilarinsufficiency.

Embolisms that may be treated, prevented and/or diagnosed with thepolynucleotides, polypeptides (including antibodies), agonists and/orantagonists of the invention include, but are not limited to, airembolisms, amniotic fluid embolisms, cholesterol embolisms, blue toesyndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms.Thrombosis include coronary thrombosis, hepatic vein thrombosis, retinalvein occlusion, carotid artery thrombosis, sinus thrombosis,Wallenberg's syndrome, and thrombophlebitis.

Ischemia that may be treated, prevented and/or diagnosed with thepolynucleotides, polypeptides (including antibodies), agonists and/orantagonists of the invention includes, but are not limited to, cerebralischemia, ischemic colitis, compartment syndromes, anterior compartmentsyndrome, myocardial ischemia, reperfusion injuries, and peripheral limbischemia. Vasculitis includes aortitis, arteritis, Behcet's Syndrome,Churg-Strauss Syndrome, mucocutaneous lymph node syndrome,thromboangiitis obliterans, hypersensitivity vasculitis,Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener'sgranulomatosis.

TIMP-4 polypeptides may be administered using any method known in theart, including, but not limited to, gene therapy (e.g., via techniquesknown in the art utilizing adenovirus vectors), gene gun, direct needleinjection at the delivery site, intravenous injection, topicaladministration, catheter infusion, biolistic injectors, particleaccelerators, gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps, oral or suppositorial solid pharmaceuticalformulations, decanting or topical applications during surgery, aerosoldelivery. Such methods are known in the art. TIMP-4 polypeptides may beadministered as part of a Therapeutic, described in more detail below.Methods of delivering TIMP-4 polynucleotides are described in moredetail herein.

The naturally occurring balance between endogenous stimulators andinhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkmanet al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985);Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science221:719-725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442-447 (1987).

The present invention provides for treatment or prevention of diseasesor disorders associated with neovascularization by administration of thepolynucleotides, polypeptides, antibodies, and/or agonists of theinvention. Malignant and metastatic conditions which can be treated withthe polynucleotides, polypeptides, antibodies, and/or agonists of theinvention include, but are not limited to, malignancies, solid tumors,and cancers described herein and otherwise known in the art (for areview of such disorders, see Fishman et al., Medicine, 2d Ed., J. B.Lippincott Co., Philadelphia (1985)).

Ocular disorders associated with neovascularization which can be treatedor prevented with the TIMP-4 polynucleotides, polypeptides, antibodies,and/or agonists of the present invention include, but are not limitedto: neovascular glaucoma, diabetic retinopathy, retinoblastoma,retrolental fibroplasia, uveitis, retinopathy of prematurity maculardegeneration, corneal graft neovascularization, as well as other eyeinflammatory diseases, ocular tumors and diseases associated withchoroidal or iris neovascularization. See, e.g., reviews by Waltman etal., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al., Surv.Ophthal. 22:291-312 (1978).

In another embodiment, TIMP-4 polypeptides, polynucleotides, antibodiesand/or agonists or antagonists of the invention are used to stimulatedifferentiation and/or survival of photoreceptor cells and/or to treator prevent diseases, disorders, or conditions associated with decreasednumber, differentiation and/or survival of photoreceptor cells.

Additionally, disorders which can be treated with the TIMP-4polynucleotides, polypeptides, antibodies, agonists and/or antagonist ofthe present invention include, but are not limited to, hemangioma,arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayedwound healing, granulations, hemophilic joints, hypertrophic scars,nonunion fractures, Osler-Weber syndrome, pyogenic granuloma,scleroderma, trachoma, and vascular adhesions.

In further embodiments, the TIMP-4 polynucleotides, polypeptides,antibodies, agonists, and/or antagonists of the inveniton, are used topromote wound healing.

In alternative embodiments, TIMP-4 polynucleotides, polypeptides,antibodies (e.g., antagonistic anti-TIMP-4 antibodies) and/orantagonists of the invention, are useful in the treatment of disordersin which stimulation of new blood vessel development would amelioratethe disorder. Such disorders include, but are not limited to, heartfailure, angina, blood vessel (e.g. coronary artery) blockage andischemia, including critical limb ischemia and refractory myocardialischemia. Antagonistic TIMP-4 polynucleotides of the invention can bedelivered to individuals to using gene therapy techniques and materialsdescribed herein or otherwise known in the art.

As a result of the ability to stimulate vascular endothelial cellgrowth, TIMP-4 antagonists of the invention, such as for example,antagonistic anti-TIMP-4 antibodies, may be employed in treatment forstimulating re-vascularization of ischemic tissues due to variousdisease conditions such as thrombosis, arteriosclerosis, and othercardiovascular conditions. The polypeptides, polynucleotides,antibodies, agonists and/or antagonists of the present invention mayalso be employed to stimulate angiogenesis and limb regeneration, asdiscussed herein.

TIMP-4 polynucleotides, polypeptides, antibodies (e.g., agonisticanti-TIMP-4 antibodies), and/or agonists can be used to inhibit MMPmediated extracellular matrix degradation or alternativelydifferentiate, proliferate, and attract cells, and thereby lead to theregeneration of tissues. (See, Science 276:59-87 (1997).) Theregeneration of tissues could be used to repair, replace, or protecttissue damaged by congenital defects, trauma (wounds, bums, incisions,or ulcers), age, disease (e.g. osteoporosis, osteocarthritis,periodontal disease, liver failure), surgery, including cosmetic plasticsurgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention includeorgans (e.g., pancreas, liver, heart, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac), vasculature(including vascular and lymphatics), nervous, hematopoietic, andskeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,regeneration occurs without or decreased scarring. Regeneration also mayinclude angiogenesis.

Moreover, TIMP-4 polynucleotides, polypeptides, antibodies, and agonistsor antagonists of the invention may increase regeneration of tissuesdifficult to heal. For example, increased tendon/ligament regenerationwould quicken recovery time after damage. TIMP-4 polynucleotides,polypeptides, antibodies, and agonists or antagonists of the presentinvention could also be used prophylactically in an effort to avoiddamage. Specific diseases that could be treated or prevented include oftendinitis, carpal tunnel syndrome, and other tendon or ligamentdefects. A further example of tissue regeneration of non-healing woundsincludes pressure ulcers, ulcers associated with vascular insufficiency,surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated according tothe present invention by using TIMP-4 polynucleotides, polypeptides,antibodies, agonists and/or antagonists to, for example, proliferate anddifferentiate nerve cells. Diseases that could be treated or preventedusing this method include, but are not limited to, central andperipheral nervous system diseases, neuropathies, or mechanical andtraumatic disorders (e.g., spinal cord disorders, head trauma,cerebrovascular disease, and stoke). Specifically, diseases associatedwith peripheral nerve injuries, peripheral neuropathy (e.g., resultingfrom chemotherapy or other medical therapies), localized neuropathies,and central nervous system diseases (e.g., Alzheimer's disease,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome), could all be treated using theTIMP-4 polynucleotides, polypeptides, antibodies, and agonists orantagonists of the invention.

Among the other diseases which TIMP-4 may be employed to treat includesalveolitis, asthma, psoriasis, glomerulosclerosis, and septic shocksince MMP's are involved in the tissue invasiveness of some parasites.

An effective amount of the TIMP-4 polynucleotides, polypeptides,antibodies and/or agonists or antagonists can be administered in vitro,ex vivo, or in vivo using techniques and compositions described hereindescribed herein (e.g., in the section entitled Therapeutic/ProphylacticAdministration and Composition) or otherwise known in the art. Byadministration of an “effective amount” of TIMP-4 polynucleotides,polypeptides, antibodies and/or agonists or antagonists is intended anamount of the compound that is sufficient to enhance or inhibit acellular response to one or more metalloproteinases. In particular, byadministration of an “effective amount” of an agonist or antagonists isintended an amount effective to enhance or inhibit TIMP-4polynucleotides, polypeptides, antibodies and/or agonists or antagonistsmediated metalloproteinase actiivity. One of ordinary skill willappreciate that effective amounts of an agonist or antagonist can bedetermined empirically and may be employed in pure form or inpharmaceutically acceptable salt, ester or pro-drug form. The agonist orantagonist may be administered in compositions in combination with oneor more pharmaceutically acceptable excipients.

It will be understood that, when administered to a human patient, thetotal daily usage of the compounds and compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgement. The specific therapeutically effective doselevel for any particular patient will depend upon factors well known inthe medical arts.

As a general proposition, the total pharmaceutically effective amount ofa TIMP-4 polypeptide administered parenterally per dose will be in therange of about 1 μg/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the TIMP-4 polypeptides are typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed.

Pharmaceutical compositions containing the TIMP-4 polypeptides of theinvention may be administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, drops or transdermal patch), bucally, or as an oralor nasal spray. By “pharmaceutically acceptable carrier” is meant anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrastemal, subcutaneous andintraarticular injection and infusion.

The pharmaceutical compositions may be administered in a convenientmanner such as by the topical, intravenous, intra-articular,intra-tumor, intraperitoneal, intramuscular, subcutaneous, intranasal orintradermal routes. The pharmaceutical compositions are administered inan amount which is effective for treating and/or prophylaxis of thespecific indication. In general, the pharmaceutical compositions areadministered in an amount of at least about 10 micrograms/kg body weightand in most cases they will be administered in an amount not in excessof about 8 mg/Kg body weight per day and preferably the dosage is fromabout 10 micrograms/kg to about 1 mg/kg body weight daily, taking intoaccount the routes of administration, symptoms, etc.

The compositions of the invention may be administered alone or incombination with other therapeutic agents, including but not limited to,chemotherapeutic agents, anti-angiogenic agents, angiogenic agents,anti-opportunistic infection agents, antivirals, antibiotics, steroidaland non-steroidal anti-inflammatories, immunosuppressants, conventionalimmunotherapeutic agents and cytokines. Combinations may be administeredeither concomitantly, e.g., as an admixture, separately butsimultaneously or concurrently; or sequentially. This includespresentations in which the combined agents are administered together asa therapeutic mixture, and also procedures in which the combined agentsare administered separately but simultaneously, e.g., as throughseparate intravenous lines into the same individual. Administration “incombination” further includes the separate administration of one of thecompounds or agents given first, followed by the second.

In one embodiment, the compositions of the invention are administered incombination with a member of the TNF family. TNF, TNF-related orTNF-like molecules that may be administered with the compositions of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, CD27L, CD30L, CD40L, 4-1BBL,DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328),AIM-I (International Publication No. WO 97/33899), AIM-II (InternationalPublication No. WO 97/34911), APRIL, endokine-alpha (InternationalPublication No. WO 98/07880), TR6 (International Publication No. WO98/30694), OPG, and neutrokine-alpha (International applicationpublication number WO 98/18921), TWEAK, OX40, and nerve growth factor(NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2(International application publication number WO 96/34095), DR3(International Publication No. WO 97/33904), DR4 (Internationalapplication publication number WO 98/32856), TR5 (Internationalapplication publication number WO 98/30693), TR7 (Internationalapplication publication number WO 98/41629), TRANK, TR9 (Internationalapplication publication number WO 98/56892), TRIO (Internationalapplication publication number WO 98/54202),312C2 (Internationalapplication publication number WO 98/06842), and TR12.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

In specific embodiments, compositions of the invention are administeredin combination with immunosuppressants. Immunosuppressants preparationsthat may be administered with the compositions of the invention include,but are not limited to, ORTHOCLONE™ (OKT3), SANDIMMUNE™/NEORAL™/SANGDYA™(cyclosporin), PROGRAF™ (tacrolimus), CELLCEPT™ (mycophenolate),Azathioprine, glucorticosteroids, and RAPAMUNE™ (sirolimus). In aspecific embodiment, immunosuppressants may be used to prevent rejectionof organ or bone marrow transplantation.

In certain embodiments, compositions of the invention are administeredin combination with antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the compositionsof the invention, include, but are not limited to, RETROVIR™(zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC),ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, CRIXIVAN™ (indinavir),NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith compositions of the invention to treat AIDS and/or to prevent ortreat HIV infection.

In other embodiments, compositions of the invention may be administeredin combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe compositions of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, compositions of theinvention are used in any combination withTRINETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat or prevent an opportunisticPneumocystis carinii pneumonia infection. In another specificembodiment, compositions of the invention are used in any combinationwith ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ toprophylactically treat or prevent an opportunistic Mycobacterium aviumcomplex infection. In another specific embodiment, compositions of theinvention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™,and/or AZITHROMYCIN™ to prophylactically treat or prevent anopportunistic Mycobacterium tuberculosis infection. In another specificembodiment, compositions of the invention are used in any combinationwith GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylacticallytreat or prevent an opportunistic cytomegalovirus infection. In anotherspecific embodiment, compositions of the invention are used in anycombination with FLUCONAZOLE™, ITRFRACONAZOLE™, and/or KETOCONAZOLE™ toprophylactically treat or prevent an opportunistic fungal infection. Inanother specific embodiment, compositions of the invention are used inany combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylacticallytreat or prevent an opportunistic herpes simplex virus type I and/ortype II infection. In another specific embodiment, compositions of theinvention are used in any combination with PYRIMETHAMINE™ and/orLEUCOVORIN™ to prophylactically treat or prevent an opportunisticToxoplasma gondii infection. In another specific embodiment,compositions of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent anopportunistic bacterial infection.

In a further embodiment, the compositions of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the compositions of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine

In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, amoxicillin, aminoglycosides, beta-lactam(glycopeptide), beta-lactamases, Clindamycin, chloramphenicol,cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin,fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim,trimethoprim-sulfamthoxazole, and vancomycin.

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the compositionsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compostions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the compositions of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophospharnide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, compositions of the invention are administeredin combination with CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone) or any combination of the components of CHOP. In anotherembodiment, compositions of the invention are administered incombination with Rituximab. In a further embodiment, compositions of theinvention are administered with Rituxmab and CHOP, or Rituxmab and anycombination of the components of CHOP.

In an additional embodiment, the compositions of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, GM-CSF, G-CSF, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12,IL13, IL15, anti-CD40, CD40L, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha,and TNF-beta. In another embodiment, compositions of the invention maybe administered with any interleukin, including, but not limited to,IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, and IL-21. In a preferred embodiment, the compositions of theinvention are administered in combination with TNF-alpha. In anotherpreferred embodiment, the compositions of the invention are administeredin combination with IFN-alpha.

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-angiogenic agent.Anti-angiogenic agents that may be administered with the compositions ofthe invention include, but are not limited to, Angiostatin (Entremed,Rockville, Md.), Troponin-1 (Boston Life Sciences, Boston, Ma.),anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel(Taxol), Suramin, Tissue Inhibitor of Metalloproteinase-1, TissueInhibitor of Metalloproteinase-2, VEGI, Plasminogen ActivatorInhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of thelighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium,molybdenum, tungsten, titanium, niobium, and tantalum species. Suchtransition metal species may form transition metal complexes. Suitablecomplexes of the above-mentioned transition metal species include oxotransition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate and orthovanadate complexes such as, forexample, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude, but are not limited to, platelet factor 4; protamine sulphate;sulphated chitin derivatives (prepared from queen crab shells), (Murataet al., Cancer Res. 51:22-26, 1991); Sulphated PolysaccharidePeptidoglycan Complex (SP-PG) (the function of this compound may beenhanced by the presence of steroids such as estrogen, and tamoxifencitrate); Staurosporine; modulators of matrix metabolism, including forexample, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline,Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al.,Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”;(Takeuchi et al., Agents Actions 36:312-316, 1992); andmetalloproteinase inhibitors such as BB94.

Additional anti-angiogenic factors that may also be utilized within thecontext of the present invention include Thalidomide, (Celgene, Warren,N.J.); Angiostatic steroid; AGM-1470 (H. Brem and J. Folkman J Pediatr.Surg. 28:445-51 (1993)); an integrin alpha v beta 3 antagonist (C.Storgard et al., J Clin. Invest. 103:47-54 (1999));carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National CancerInstitute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXiGENE, Boston,Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.);TNP-470, (Tap Pharmaceuticals, Deerfield, Ill.); ZD-0101 AstraZeneca(London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251(PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin;Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide(Somatostatin); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat(AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (Nolvadex);Tazarotene; Tetrathiomolybdate; Xeloda (Capecitabine); and5-Fluorouracil.

Anti-angiogenic agents that may be administed in combination with thecompounds of the invention may work through a variety of mechanismsincluding, but not limited to, inhibiting proteolysis of theextracellular matrix, blocking the function of endothelialcell-extracellular matrix adhesion molecules, by antagonizing thefunction of angiogenesis inducers such as growth factors, and inhibitingintegrin receptors expressed on proliferating endothelial cells.Examples of anti-angiogenic inhibitors that interfere with extracellularmatrix proteolysis and which may be administered in combination with thecompositons of the invention include, but are not lmited to, AG-3340(Agouron, La Jolla, Calif.), BAY-12-9566 (Bayer, West Haven, Conn.),BMS-275291 (Bristol Myers Squibb, Princeton, N.J.), CGS-27032A(Novartis, East Hanover, N.J.), Marimastat (British Biotech, Oxford,UK), and Metastat (Aeterna, St-Foy, Quebec). Examples of anti-angiogenicinhibitors that act by blocking the function of endothelialcell-extracellular matrix adhesion molecules and which may beadministered in combination with the compositons of the inventioninclude, but are not lmited to, EMD-121974 (Merck KcgaA Darmstadt,Germany) and Vitaxin (Ixsys, La Jolla, Calif./Medimmune, Gaithersburg,Md.). Examples of anti-angiogenic agents that act by directlyantagonizing or inhibiting angiogenesis inducers and which may beadministered in combination with the compositons of the inventioninclude, but are not lmited to, Angiozyme (Ribozyme, Boulder, Colo.),Anti-VEGF antibody (Genentech, S. San Francisco, Calif.),PTK-787/ZK-225846 (Novartis, Basel, Switzerland), SU-101 (Sugen, S. SanFrancisco, Calif.), SU-5416 (Sugen/Pharmacia Upjohn, Bridgewater, N.J.),and SU-6668 (Sugen). Other anti-angiogenic agents act to indirectlyinhibit angiogenesis. Examples of indirect inhibitors of angiogenesiswhich may be administered in combination with the compositons of theinvention include, but are not Imited to, IM-862 (Cytran, Kirkland,Wash.), Interferon-alpha, IL-12 (Roche, Nutley, N.J.), and Pentosanpolysulfate (Georgetown University, Washington, D.C.).

In particular embodiments, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of an autoimmune disease,such as for example, an autoimmune disease described herein.

In a particular embodiment, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of arthritis. In a moreparticular embodiment, the use of compositions of the invention incombination with anti-angiogenic agents is contemplated for thetreatment, prevention, and/or amelioration of rheumatoid arthritis.

In an additional embodiment, the compositions of the invention areadministered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the compositions of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PlGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PlGF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B-186 (VEGF-B186), as disclosed in InternationalPublication Number WO 96/26736; Vascular Endothelial Growth Factor-D(VEGF-D), as disclosed in International Publication Number WO 98/02543;Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/07832; and Vascular EndothelialGrowth Factor-E (VEGF-E), as disclosed in German Patent NumberDE19639601. The above mentioned references are incorporated herein byreference herein.

In an additional embodiment, the compositions of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors that may be administered with the compositions of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

In additional embodiments, the compositions of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

This invention is also related to the use of the human TIMP-4 gene aspart of a diagnostic assay for detecting diseases or susceptibility todiseases related to the presence of mutated human TIMP-4.

Individuals carrying mutations in the human TIMP-4 gene may be detectedat the DNA level by a variety of techniques. Nucleic acids for diagnosismay be obtained from a patient's cells, such as from blood, urine,saliva, tissue biopsy and autopsy material. The genomic DNA may be useddirectly for detection or may be amplified enzymatically by using PCR(Saiki et al., Nature, 324:163-166 (1986)) prior to analysis. RNA orcDNA may also be used for the same purpose. As an example, PCR primerscomplementary to the nucleic acid encoding human TIMP-4 can be used toidentify and analyze human TIMP-4 mutations. For example, deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to radiolabeled human TIMP-4 RNA oralternatively, radiolabeled human TIMP-4 antisense DNA sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase A digestion or by differences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of human TIMP-4 protein in various tissues since anover-expression of the proteins compared to normal control tissuesamples may detect the presence of a disease or susceptibility to adisease regulated by human TIMP-4. Assays used to detect levels of humanTIMP-4 protein in a sample derived from a host are well-known to thoseof skill in the art and include radioimmunoassays, competitive-bindingassays, Western Blot analysis, ELISA assays and “sandwich” assay. AnELISA assay (Coligan, et al., Current Protocols in Immunology, 1(2),Chapter 6, (1991)) initially comprises preparing an antibody specific tothe human TIMP-4 antigen, preferably a monoclonal antibody. In additiona reporter antibody is prepared against the monoclonal antibody. To thereporter antibody is attached a detectable reagent such asradioactivity, fluorescence or, in this example, a horseradishperoxidase enzyme. A sample is removed from a host and incubated on asolid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein like BSA. Next, the monoclonalantibody is incubated in the dish during which time the monoclonalantibodies attach to any human TIMP-4 proteins attached to thepolystyrene dish. All unbound monoclonal antibody is washed out withbuffer. The reporter antibody linked to horseradish peroxidase is nowplaced in the dish resulting in binding of the reporter antibody to anymonoclonal antibody bound to human TIMP-4. Unattached reporter antibodyis then washed out. Peroxidase substrates are then added to the dish andthe amount of color developed in a given time period is a measurement ofthe amount of human TIMP-4 protein present in a given volume of patientsample when compared against a standard curve.

A competition assay may be employed wherein antibodies specific to humanTIMP-4 are attached to a solid support and labeled human TIMP-4 and asample derived from the host are passed over the solid support and theamount of label detected, for example by liquid scintillationchromatography, can be correlated to a quantity of human TIMP-4 in thesample.

A “sandwich” assay is similar to an ELISA assay. In a “sandwich” assayhuman TIMP-4 is passed over a solid support and binds to antibodyattached to a solid support. A second antibody is then bound to thehuman TIMP-4. A third antibody which is labeled and specific to thesecond antibody is then passed over the solid support and binds to thesecond antibody and an amount can then be quantitated.

This invention also provides a method of screening compounds to identifythose which are agonists or antagonists to be human TIMP-4 polypeptide.An example of such a method comprises obtaining mammalian tissuecomprising an extra-cellular matrix, for example, bovine radiocarpaljoints. The articular cartilage is cut into smaller disks and labeledwith ³⁵S-sodium sulfate (10 micro Ci/ml) in DMEM for a sufficient timefor the cartilage to incorporate the labeled Sodium sulfate. An MMP, forexample, stromelysin, or IL1 or TNF is then added to the cartilage disksunder appropriate conditions such that tissue breakdown would normallyoccur. Human TIMP-4 and the compounds to be screened are then added tothe reaction mixture for a sufficient time for the MMP to normally breakdown the cartilage disks. The supernatant, which is the media outsidethe cartilage disks, is then collected and radioactivity is counted by aliquid scintillation counter. The percentage of ³⁵S released into themedia is then calculated. This release of ³⁵S-GAG is representative ofthe proteoglycan pool in the extracellular matrix of cartilage, andreflects proteoglycan degradation by the MMP. The amount of ³⁵S-GAG, asdetermined by liquid scintillation chromatography, is then compared to acontrol assay done in the absence of the compound to be screened and theability of the compound to agonize or antagonize the action of humanTIMP-4 may then be determined.

Examples of potential human TIMP-4 antagonists, in addition to thoseidentified above, include an antibody, or in some cases, anoligonucleotide, which binds to the polypeptide. Alternatively, apotential antagonist may be a mutated form of human TIMP-4, whichrecognizes natural substrates, but is inactive, and thereby prevent theaction of human TIMP-4.

Potential human TIMP-4 antagonists also include antisense constructsprepared using antisense technology. Antisense technology can be used tocontrol gene expression through triple-helix formation or antisense DNAor RNA, both of which methods are based on binding of a polynucleotideto DNA or RNA. For example, the 5′ coding portion of the polynucleotidesequence, which encodes for the mature polypeptides of the presentinvention, is used to design an antisense RNA oligonucleotide of fromabout 10 to 40 base pairs in length. A DNA oligonucleotide is designedto be complementary to a region of the gene involved in transcription(triple helix—see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney etal, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360(1991)), thereby preventing transcription and the production of humanTIMP-4. The antisense RNA oligonucleotide hybridizes to the mRNA in vivoand blocks translation of the mRNA molecule into the human TIMP-4(antisense—Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of human TIMP-4.

Another potential human TIMP-4 antagonist is a small molecule whichbinds to and occupies the active site of the human TIMP-4 therebypreventing human TIMP-4 from interacting with MMP's such that normalbiological activity is prevented. Examples of small molecules includebut are not limited to small peptides or peptide-like molecules, forexample a peptide-bonded molecule.

The human TIMP-4 antagonists may be employed for tissue repair andremodeling, for example, where destruction of scar tissue is desired. Insome situations, enhanced connective tissue turnover or remodeling maybe desirable, e.g. in resorption of scar tissue; in uterine involutionpost-partum; in remodeling of fibrotic deposits in the lung, liver orjoints. To appropriately control turnover of extra-cellular matrixproteins in these situations would require a balance between the MMP'sand human TIMP-4 to appropriately control degradation.

The polypeptides and agonists or antagonists that are also polypeptidesmay be employed in accordance with the present invention by expressionof such polypeptides in vivo, which is often referred to as “genetherapy.”

Gene Therapy

The invention also encompasses gene therapy methods for treating orpreventing disorders, diseases and conditions, such as, for examplerestenosis. Vectors and techniques described herein (e.g, below or inthe Antibody section of the application) or known in the art may beroutinely applied or modified for such therapy. Gene therapy methodsrelate to the introduction of nucleic acid (DNA, RNA and antisense DNAor RNA) sequence of the invention into an animal to achieve expressionof the TIMP-4 polypeptide of the present invention. This method requiresa polynucleotide which codes for a TIMP-4 polypeptide operatively linkedto a promoter and any other genetic elements necessary for theexpression of the polypeptide by the target tissue. Such gene therapyand delivery techniques are known in the art, see, for example,WO90/11092, which is herein incorporated by reference.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to aTIMP-4 polynucleotide ex vivo, with the engineered cells then beingprovided to a patient to be treated with the polypeptide. Such methodsare well-known in the art. For example, see Belldegrun, A., et al., J.Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., CancerResearch 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995);Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato,L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al.,Gene Therapy 4:1246-1255 (1997); and Zhang, J. -F. et al., Cancer GeneTherapy 3: 31-38 (1996)), which are herein incorporated by reference. Inone embodiment, the cells which are engineered are arterial cells. Thearterial cells may be reintroduced into the patient through directinjection to the artery, the tissues surrounding the artery, or throughcatheter injection.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current Opinion in Genetics and Development 3:499-503 present a reviewof adenovirus-based gene therapy. Bout et al., 1994, Human Gene Therapy5:3-10 demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al., 1991,Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT PublicationWO94/12649; and Wang, et al., 1995, Gene Therapy 2:775-783.

As discussed in more detail herein (e.g., below and in the antibodysection of the application), the TIMP-4 polynucleotide constructs can bedelivered by any method that delivers injectable materials to the cellsof an animal, such as, injection into the interstitial space of tissues(heart, muscle, skin, lung, liver, and the like). The TIMP-4polynucleotide constructs may be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

In one embodiment, the TIMP-4 polynucleotide is delivered as a nakedpolynucleotide. The term “naked” polynucleotide, DNA or RNA refers tosequences that are free from any delivery vehicle that acts to assist,promote or facilitate entry into the cell, including viral sequences,viral particles, liposome formulations, lipofectin or precipitatingagents and the like. However, the TIMP-4 polynucleotides can also bedelivered in liposome formulations and lipofectin formulations and thelike can be prepared by methods well known to those skilled in the art.Such methods are described, for example, in U.S. Pat. Nos. 5,593,972,5,589,466, and 5,580,859, which are herein incorporated by reference.

The TIMP-4 polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication.Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSGavailable from Stratagene; pSVK3, pBPV, pMSG and pSVL available fromPharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

Any strong promoter known to those skilled in the art can be used fordriving the expression of TIMP-4 polynucleotide sequence. Suitablepromoters include adenoviral promoters, such as the adenoviral majorlate promoter; or heterologous promoters, such as the cytomegalovirus(CMV) promoter; the respiratory syncytial virus (RSV) promoter;inducible promoters, such as the MMT promoter, the metallothioneinpromoter; heat shock promoters; the albumin promoter; the ApoAIpromoter; human globin promoters; viral thymidine kinase promoters, suchas the Herpes Simplex thymidine kinase promoter; retroviral LTRs; theb-actin promoter; and human growth hormone promoters. The promoter alsomay be the native promoter for TIMP-4.

Unlike other gene therapy techniques, one major advantage of introducingnaked nucleic acid sequences into target cells is the transitory natureof the polynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The TIMP-4 polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellular,fluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked nucleic acid sequence injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 mg/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked TIMP-4 DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such asviral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

In certain embodiments, the TIMP-4 polynucleotide constructs arecomplexed in a liposome preparation. Liposomal preparations for use inthe instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA(1987) 84:7413-7416, which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which isherein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which is hereinincorporated by reference), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated byreference). Other commercially available liposomes include transfectace(DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication No. WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., P. Felgneret al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology (1983), 101:512-527, which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca²⁺-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilsonet al., Cell (1979) 17:77); ether injection (Deamer, D. and Bangham, A.,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); detergent dialysis (Enoch, H. and Strittmatter, P.,Proc. Natl. Acad. Sci. USA (1979) 76:145); and reverse-phase evaporation(REV) (Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka, F. andPapahadjopoulos, D., Proc. Natl. Acad. Sci. USA (1978) 75:145;Schaefer-Ridder et al., Science (1982) 215:166), which are hereinincorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10. Preferably, the ration will be from about 5:1 to about 1:5.More preferably, the ration will be about 3:1 to about 1:3. Still morepreferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication no. WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication no. WO 94/9469 (which areherein incorporated by reference) provide methods for deliveringDNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, usinga retroviral particle containing RNA which comprises a sequence encodingTIMP-4. Retroviruses from which the retroviral plasmid vectors may bederived include, but are not limited to, Moloney Murine Leukemia Virus,spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avianleukosis virus, gibbon ape leukemia virus, human immunodeficiency virus,Myeloproliferative Sarcoma Virus, and mammary tumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, Ψ-2,Ψ-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990),which is incorporated herein by reference in its entirety. The vectormay transduce the packaging cells through any means known in the art.Such means include, but are not limited to, electroporation, the use ofliposomes, and CaPO₄ precipitation. In one alternative, the retroviralplasmid vector may be encapsulated into a liposome, or coupled to alipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding TIMP-4. Such retroviral vectorparticles then may be employed, to transduce eukaryotic cells, either invitro or in vivo. The transduced eukaryotic cells will express TIMP-4.

In certain other embodiments, cells are engineered, ex vivo or in vivo,with TIMP-4 polynucleotide contained in an adenovirus vector. Adenoviruscan be manipulated such that it encodes and expresses TIMP-4, and at thesame time is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. Adenovirus expression is achieved withoutintegration of the viral DNA into the host cell chromosome, therebyalleviating concerns about insertional mutagenesis. Furthermore,adenoviruses have been used as live enteric vaccines for many years withan excellent safety profile (Schwartz, A. R. et al. (1974) Am. Rev.Respir. Dis.109:233-238). Finally, adenovirus mediated gene transfer hasbeen demonstrated in a number of instances including transfer ofalpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M.A. et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell68:143-155). Furthermore, extensive studies to attempt to establishadenovirus as a causative agent in human cancer were uniformly negative(Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).

In cases where an adenovirus is used as an expression vector, the TIMP-4coding sequence of interest may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingthe TIMP-4 molecule in infected hosts. (e.g., see Logan & Shenk, 1984,Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals mayalso be required for efficient translation of inserted antibody codingsequences. These signals include the ATG initiation codon and adjacentsequences. Furthermore, the initiation codon must be in phase with thereading frame of the desired coding sequence to ensure translation ofthe entire insert. These exogenous translational control signals andinitiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc. (see Bittner et al., 1987, Methods in Enzymol.153:51-544).

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300;U.S. Pat. No. 5,436,146).

Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992);Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al.,Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692(1993); U.S. Pat. No. 6,040,174, U.S. Pat. No. 6,013,638, and U.S. Pat.No. 5,652,224, each of which are herein incorporated by reference in itsentirety. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the El region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention. (Seee.g., U.S. Pat. Nos. 6,040,174 and 6,013,638, the contents of each ofwhich are incorporated by reference in its entirety).

Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or invivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol.158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377,the contents of each of which are herein incorporated by reference inits entirety.

For example, an appropriate AAV vector for use in the present inventionwill include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The TIMP-4 polynucleotideconstruct is inserted into the AAV vector using standard cloningmethods, such as those found in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAVvector is then transfected into packaging cells which are infected witha helper virus, using any standard technique, including lipofection,electroporation, calcium phosphate precipitation, etc. Appropriatehelper viruses include adenoviruses, cytomegaloviruses, vacciniaviruses, or herpes viruses. Once the packaging cells are transfected andinfected, they will produce infectious AAV viral particles which containthe TIMP-4 polynucleotide construct. These viral particles are then usedto transduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the TIMP-4 polynucleotide construct integrated intoits genome, and will express TIMP-4.

Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding TIMP-4) via homologous recombination (see, e.g., U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No.WO 96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989).This method involves the activation of a gene which is present in thetarget cells, but which is not normally expressed in the cells, or isexpressed at a lower level than desired.

Polynucleotide constructs are made, using standard techniques known inthe art, which contain the promoter with targeting sequences flankingthe promoter. Suitable promoters are described herein. The targetingsequence is sufficiently complementary to an endogenous sequence topermit homologous recombination of the promoter-targeting sequence withthe endogenous sequence. The targeting sequence will be sufficientlynear the 5′ end of the TIMP-4 desired endogenous polynucleotide sequenceso the promoter will be operably linked to the endogenous sequence uponhomologous recombination.

The promoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

The promoter-targeting sequence construct is delivered to the cells,either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous TIMP-4 sequence is placedunder the control of the promoter. The promoter then drives theexpression of the endogenous TIMP-4 sequence.

The polynucleotides encoding TIMP-4 may be administered along with otherpolynucleotides encoding an angiogenic protein. Examples of angiogenicproteins include, but are not limited to, acidic and basic fibroblastgrowth factors, VEGF-1, VEGF-2, VEGF-3, epidermal growth factor alphaand beta, platelet-derived endothelial cell growth factor,platelet-derived growth factor, tumor necrosis factor alpha, hepatocytegrowth factor, insulin like growth factor, colony stimulating factor,macrophage colony stimulating factor, granulocyte/macrophage colonystimulating factor, and nitric oxide synthase.

Preferably, the polynucleotide encoding TIMP-4 contains a secretorysignal sequence that facilitates secretion of the protein. Typically,the signal sequence is positioned in the coding region of thepolynucleotide to be expressed towards or at the 5′ end of the codingregion. The signal sequence may be homologous or heterologous to thepolynucleotide of interest and may be homologous or heterologous to thecells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotidesconstructs can be used so long as the mode results in the expression ofone or more molecules in an amount sufficient to provide a therapeuticeffect. This includes direct needle injection, systemic injection,catheter infusion, biolistic injectors, particle accelerators (i.e.,“gene guns”), gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps (e.g., Alza minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, and decanting ortopical applications during surgery. For example, direct injection ofnaked calcium phosphate-precipitated plasmid into rat liver and ratspleen or a protein-coated plasmid into the portal vein has resulted ingene expression of the foreign gene in the rat livers (Kaneda et al.,Science 243:375 (1989)).

A preferred method of local administration is by direct injection.Preferably, a recombinant molecule of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of arteries. Administration of a compositionlocally within the area of arteries refers to injecting the compositioncentimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotideconstruct of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery and the polynucleotide constructcan be coated on the surface of tissue inside the wound or the constructcan be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian.

Therapeutic compositions of the present invention can be administered toany animal, preferably to mammals and birds. Preferred mammals includehumans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs,with humans being particularly preferred.

Kits

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepharmaceutical compositions may be employed in conjunction with othertherapeutic compounds.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clones to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press,New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice may be used to express humanized antibodies to immunogenicpolypeptide products of this invention.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 microgram of plasmid or DNA fragment is used withabout 2 units of enzyme in about 20 microliters of buffer solution. Forthe purpose of isolating DNA fragments for plasmid construction,typically 5 to 50 micrograms of DNA are digested with 20 to 250 units ofenzyme in a larger volume. Appropriate buffers and substrate amounts forparticular restriction enzymes are specified by the manufacturer.Incubation times of about 1 hour at 37° C. are ordinarily used, but mayvary in accordance with the supplier's instructions. After digestion thereaction is electrophoresed directly on a polyacrylamide gel to isolatethe desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units to T4 DNA ligase (“ligase”)per 0.5 micrograms of approximately equimolar amounts of the DNAfragments to be ligated.

Unless otherwise stated, transformation was performed as described inthe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, and Examples of this specification is hereby incorporatedby reference in its entirety.

In addition, the entire disclosure, including the specifications andsequence listings, of related U.S. application Ser. No. 09/387,525,filed Sep. 1, 1999; Ser. No. 08/463,261, filed Jun. 5, 1995; No.60/217,419, filed Jul. 11, 2000; No. 60/220,829, filed Jul. 26, 2000;and International Application No. PCT/US94/14498, filed Dec. 13, 1994(in English), are each hereby incorporated by reference in theirentireties.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLE 1

Bacterial Expression and Purification of Human TIMP-4

The DNA sequence encoding for human TIMP-4, ATCC #75946, is initiallyamplified using PCR oligonucleotide primers corresponding to the 5′ andsequences of the processed human TIMP-4 protein (minus the signalpeptide sequence) and the vector sequences 3′ to the TIMP-4 gene.Additional nucleotides corresponding to human TIMP-4 were added to the5′ and 3′ sequences respectively. The 5′ oligonucleotide primer has thesequence 5′ GCCAGAGGATCCTGCAGCTGCGCCCCGGCGCAC 3′ (SEQ ID NO:3) containsa BamHI restriction enzyme site followed by 21 nucleotides of humanTIMP-4 coding sequence starting from the presumed terminal amino acid ofthe processed protein codon. The 3′ sequence 5°C.GGCTTCTAGAACTAGGGCTGAACGATGTCAAC 3′ (SEQ ID NO:4) contains an XbaIsite and is followed by 18 nucleotides of human TIMP-4. The restrictionenzyme sites correspond to the restriction enzyme sites on the bacterialexpression vector pQE-9 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth,Calif., 91311). pQE-9 encodes antibiotic resistance (Amp^(r)), abacterial origin of replication (ori), an IPTG-regulatable promoteroperator (P/O), a ribosome binding site (RBS), a 6-His tag andrestriction enzyme sites. pQE-9 was then digested with BamH1 and XbaI.The amplified sequences were ligated into pQE-9 and were inserted inframe with the sequence encoding for the histidine tag and the RBS. Theligation mixture was then used to transform E. coli strain m15/pREP4available from Qiagen by the procedure described in Sambrook, J. et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press,(1989). m15/pREP4 contains multiple copies of the plasmid pREP4, whichexpresses the lacI repressor and also confers kanamycin resistance(Kan^(r)). Transformants are identified by their ability to grow on LBplates and ampicillin/kanamycin resistant colonies were selected.Plasmid DNA was isolated and confirmed by restriction analysis.

Clones containing the desired constructs were grown overnight (ON) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells were grown to an optical density600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalactopyranoside”) was then added to a final concentration of 1 mM. IPTGinduces by inactivating the lacI repressor, clearing the P/O leading toincreased gene expression. Cells were grown an extra 3 to 4 hours. Cellswere then harvested by centrifugation. The cell pellet was solubilizedin the chaotropic agent 6 Molar Guanidine HCl. After clarification,solubilized human TIMP-4 was purified from this solution bychromatography on a Nickel-Chelate column under conditions that allowfor tight binding by proteins containing the 6-His tag (Hochuli, E. etal., J. Chromatography 411:177-184 (1984). Human TIMP-4 (90% pure) waseluted from the column in 6 molar guanidine HCl pH 5.0 and for thepurpose of renaturation adjusted to 3 molar guanidine HCl, 100 mM sodiumphosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione(oxidized). After incubation in this solution for 12 hours the proteinwas dialyzed to 10 mmolar sodium phosphate.

EXAMPLE 2

Expression of Recombinant Human TIMP-4 in COS cells

The expression of human TIMP-4 HA is derived from a vector pcDNAI/Amp(Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillinresistance gene, 3) E.coli replication origin, 4) CMV promoter followedby a polylinker region, a SV40 intron and polyadenylation site. A DNAfragment encoding the entire human TIMP-4 precursor and a HA tag fusedin frame to its 3′ end was cloned into the polylinker region of thevector, therefore, the recombinant protein expression is directed underthe CMV promoter. The HA tag correspond to an epitope derived from theinfluenza hemagglutinin protein as previously described (I. Wilson, H.Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lemer, 1984, Cell37, 767). The fusion of HA tag to the target protein allows easydetection of the recombinant protein with an antibody that recognizesthe HA epitope.

The plasmid construction strategy is described as follows:

The DNA sequence ATCC # 75946, encoding for human TIMP-4 was constructedby PCR using two primers: the 5′ primer 5′ GCCAGAGGATCCGCCACCATGCCTGGGAGCCCTCGGCCC 3′ (SEQ ID NO:5) contains a BamHI sitefollowed by 21 nucleotides of human TIMP-4 coding sequence starting fromthe initiation codon; the 3′ sequence 5′CGGCTTCTAGAATCAAGCGTAGTCTGGGACGTCG TATGGGTAGGGCTGAACGATGTCAAC 3′ (SEQ IDNO:6) contains complementary sequences to an XbaI site, translation stopcodon, HA tag and the last 18 nucleotides of the human TIMP-4 codingsequence (not including the stop codon). Therefore, the PCR productcontains a BamHI site, human TIMP-4 coding sequence followed by HA tagfused in frame, a translation termination stop codon next to the HA tag,and an XbaI site. The PCR amplified DNA fragment and the vector,pcDNAI/Amp, were digested with BamHI and XbaI restriction enzyme andligated. The ligation mixture was transformed into E. coli strain SURE(available from Stratagene Cloning Systems, 11099 North Torrey PinesRoad, La Jolla, Calif. 92037) the transformed culture was plated onampicillin media plates and resistant colonies were selected. PlasmidDNA was isolated from transformants and examined by restriction analysisfor the presence of the correct fragment. For expression of therecombinant human TIMP-4, COS cells were transfected with the expressionvector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press,(1989)). The expression of the human TIMP-4 HA protein was detected byradiolabelling and immunoprecipitation method (E. Harlow, D. Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,(1988)). Cells were labelled for 8 hours with ³⁵S-cysteine two days posttransfection. Culture media were then collected and cells were lysedwith detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40,0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)).Both cell lysate and culture media were precipitated with a HA specificmonoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAGEgels.

EXAMPLE 3

Cloning and Expression of TIMP-4 Using the Baculovirus Expression System

The DNA sequence encoding the full length TIMP-4 protein, ATCC # 75946,was amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ sequences of the gene:

The 5′ primer has the sequence 5′ GCCAGAGGATCCATGCCTGG GAGCCCTCGGCCC 3′(SEQ ID NO:7) and contains a BamHI restriction enzyme site (in bold)just behind the first 21 nucleotides of the TIMP-4 gene (the initiationcodon for translation “ATG” is underlined).

The 3′ primer has the sequence 5′ CGGCTTCTAGAACTAGGGCTG AACGATGTCAAC 3′(SEQ ID NO:8) and contains the cleavage site for the restrictionendonuclease XbaI and 18 nucleotides complementary to the 3′non-translated sequence of the TIMP-4 gene. The amplified sequences wereisolated from a 1% agarose gel using a commercially available kit(“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment was thendigested with the endonucleases BamHI and XbaI and then purified againon a 1% agarose gel. This fragment is designated F2.

The vector pA2 (modification of pVL941 vector, discussed below) is usedfor the expression of the TIMP-4 protein using the baculovirusexpression system (for review see: Summers, M. D. and Smith, G. E. 1987,A manual of methods for baculovirus vectors and insect cell cultureprocedures, Texas Agricultural Experimental Station Bulletin No. 1555).This expression vector contains the strong polyhedrin promoter of theAutographa califomica nuclear polyhidrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases BamHI and XbaI.The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusesthe beta-galactosidase gene from E.coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of co-transfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pRG1 such as pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid was digested with the restriction enzymes BamHI and XbaI.The DNA was then isolated from a 1% agarose gel using the commerciallyavailable kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.). This vectorDNA is designated V2.

Fragment F2 and the plasmid V2 were ligated with T4 DNA ligase. E.coliHB101 cells were then transformed and bacteria identified that containedthe plasmid (pBacTIMP-4) with the TIMP-4 gene using the enzymes BamHIand XbaI. The sequence of the cloned fragment was confirmed by DNAsequencing.

5 micrograms of the plasmid pBacTIMP-4 was co-transfected with 1.0microgram of a commercially available linearized baculovirus(“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.) using thelipofection method (Felgner et al. Proc. Natl. Acad. Sci. USA,84:7413-7417 (1987)).

One microgram of BaculoGold™ virus DNA and 5 micrograsm of the plasmidpBacTIMP-4 were mixed in a sterile well of a microtiter plate containing50 microliters of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 microliters Lipofectin plus 90microliters Grace's medium were added, mixed and incubated for 15minutes at room temperature. Then the transfection mixture was addeddrop-wise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mmtissue culture plate with 1 ml Grace's medium without serum. The platewas rocked back and forth to mix the newly added solution. The plate wasthen incubated for 5 hours at 27 degree C. After 5 hours thetransfection solution was removed from the plate and 1 ml of Grace'sinsect medium supplemented with 10% fetal calf serum was added. Theplate was put back into an incubator and cultivation continued at 27degree C. for four days.

After four days the supernatant was collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the viruses were added to the cellsand blue stained plaques were picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses was thenresuspended in an Eppendorf tube containing 200 microliters of Grace'smedium. The agar was removed by a brief centrifugation and thesupernatant containing the recombinant baculovirus was used to infectSf9 cells seeded in 35 mm dishes. Four days later the supernatants ofthese culture dishes were harvested and then stored at 4 degree C.

Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-TIMP-4 at a multiplicity of infection (MOI) of 2. Sixhours later the medium was removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 microCi of ³⁵S-methionine and 5 microCi 35S cysteine(Amersham) were added. The cells were further incubated for 16 hoursbefore they were harvested by centrifugation and the labelled proteinsvisualized by SDS-PAGE and autoradiography.

EXAMPLE 4

Expression Pattern of Human TIMP-4 in Human Tissues

20 micrograms of total RNA from each of the above tissues was denaturedand run on a 1.2% formaldehyde agarose gel and capillary blotted onto anylon filter overnight. RNA was immobilized on the filter by UVcross-linking. A random primer probe was prepared from the EcoRI-Xholinsert of the partial TIMP-4 nucleic acid sequence and used to probe theblot by overnight hybridization in Church buffer with 100 μg/mldenatured herring sperm DNA as a blocking agent. Washing was performedsequentially with 2× SSC/0.1% SDA and 0.2× SSC/0.1% SDS at 65 degreesCelsius.

EXAMPLE 5

Expression via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37 degree C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5′ and 3′ end sequencesrespectively. The 5′ primer contains an EcoRI site and the 3′ primerfurther includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

EXAMPLE 6

Production of an Antibody

a) Hybridoma Technology

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing TIMP-4 are administered to an animal to inducethe production of sera containing polyclonal antibodies. In a preferredmethod, a preparation of TIMP-4 protein is prepared and purified torender it substantially free of natural contaminants. Such a preparationis then introduced into an animal in order to produce polyclonalantisera of greater specific activity.

Monoclonal antibodies specific for protein TIMP-4 are prepared usinghybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler etal., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol.6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal(preferably a mouse) is immunized with TIMP-4 polypeptide or, morepreferably, with a secreted TIMP-4 polypeptide-expressing cell. Suchpolypeptide-expressing cells are cultured in any suitable tissue culturemedium, preferably in Earle's modified Eagle's medium supplemented with10% fetal bovine serum (inactivated at about 56° C.), and supplementedwith about 10 g/l of nonessential amino acids, about 1,000 U/ml ofpenicillin, and about 100 μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP20), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981). The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the TIMP-4 polypeptide.

Alternatively, additional antibodies capable of binding to TIMP-4polypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodywhich binds to a second antibody. In accordance with this method,protein specific antibodies are used to immunize an animal, preferably amouse. The splenocytes of such an animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify cloneswhich produce an antibody whose ability to bind to the TIMP-4protein-specific antibody can be blocked by TIMP-4. Such antibodiescomprise anti-idiotypic antibodies to the TIMP-4 protein-specificantibody and are used to immunize an animal to induce formation offurther TIMP-4 protein-specific antibodies.

For in vivo use of antibodies in humans, an antibody is “humanized”.Such antibodies can be produced using genetic constructs derived fromhybridoma cells producing the monoclonal antibodies described above.Methods for producing chimeric and humanized antibodies are known in theart and are discussed infra. (See, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature314:268 (1985).)

b) Isolation of Antibody Fragments Directed Against TIMP-4 from aLibrary of scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a library of antibody fragments which contain reactivities againstTIMP-4 to which the donor may or may not have been exposed (see e.g.,U.S. Pat. No. 5,885,793 incorporated herein by reference in itsentirety).

Rescue of the Library.

A library of scFvs is constructed from the RNA of human PBLs asdescribed in PCT publication WO 92/01047. To rescue phage displayingantibody fragments, approximately 109 E. coli harboring the phagemid areused to inoculate 50 ml of 2xTY containing 1% glucose and 100 μg/ml ofampicillin (2xTY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Fiveml of this culture is used to innoculate 50 ml of 2xTY-AMP-GLU, 2×108 TUof delta gene 3 helper (M13 delta gene III, see PCT publication WO92/01047) are added and the culture incubated at 37° C. for 45 minuteswithout shaking and then at 37° C. for 45 minutes with shaking. Theculture is centrifuged at 4000 r.p.m. for 10 min. and the pelletresuspended in 2 liters of 2xTY containing 100 jig/ml ampicillin and 50ug/ml kanamycin and grown overnight. Phage are prepared as described inPCT publication WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),resuspended in 300 ml 2xTY broth containing 100 jig ampicillin/ml and 25μg kanamycin/ml (2xTY-AMP-KAN) and grown overnight, shaking at 37° C.Phage particles are purified and concentrated from the culture medium bytwo PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBSand passed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 1013 transducing units/ml(ampicillin-resistant clones).

Panning of the Library.

Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100μg/ml or 10 μg/ml of a polypeptide of the present invention. Tubes areblocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 timesin PBS. Approximately 1013 TU of phage is applied to the tube andincubated for 30 minutes at room temperature tumbling on an over andunder turntable and then left to stand for another 1.5 hours. Tubes arewashed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage areeluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes onan under and over turntable after which the solution is immediatelyneutralized with 0.5 ml of 1.0 M Tris-HCl, pH 7.4. Phage are then usedto infect 10 ml of mid-log E. coli TG1 by incubating eluted phage withbacteria for 30 minutes at 37° C. The E. coli are then plated on TYEplates containing 1% glucose and 100 μg/ml ampicillin. The resultingbacterial library is then rescued with delta gene 3 helper phage asdescribed above to prepare phage for a subsequent round of selection.This process is then repeated for a total of 4 rounds of affinitypurification with tube-washing increased to 20 times with PBS, 0.1%Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing.

EXAMPLE 7

Adenoviral Mediated Gene Therapy

Adenoviral expression constructs were used to express human and ratTIMP-4 polypeptides in rat thoracic aorta smooth muscle cells and humanand porcine coronary artery smooth muscle cells in vitro, as well as inan in vivo rat model of carotid artery balloon injury.

Adenoviral Constructions:

The human cDNA sequence encoding the full length TIMP-4 protein,contained in ATCC Deposit No. 75946, was isolated by PCR from cloneHGFAM58 using oligonucleotide primers corresponding to the 5′ and 3′sequence of the gene. The 5′ primer has the sequence 5′CCGGAATTCCACCATGCCTGGGAGCCCTCG 3′ (SEQ ID NO:9) and contains an EcoRIrestriction enzyme site behind the first 17 nucleotides of the TIMP-4gene. The 3′ primer has the sequence 5′ ATCTTTGGTACCTTTCTAGAACTAGGGCTG3′ (SEQ ID NO:10) and contains a XbaI restriction enzyme site and 14nucleotides complementary to the 3′ non-translated sequence of theTIMP-4 gene.

The rat cDNA sequence encoding the full length TIMP-4 protein wasisolated by RT-PCR using RNA extracted from rat aortic smooth musclecells. Briefly, 2 μg of total RNA was reverse transcribed in 20 μl finalreaction volume in the presence of the four dNTPs, 100 pmol of pdN6(Boehringer Mannheim, Ingelheim, Germany) and 200IU of reversetranscriptase (Superscript, Promega, France). The reaction was performedat 37° C. for 75 min. Then, the enzyme was denatured at 90° C. for 5min. 2 μl of the reaction was used as a template for PCR using specificoligonucleotide promoters for the rat TIMP-4. The sense primer has thesequence 5′ CCGGAATTCCACCATGCCCTGGAGTCCC3′ (SEQ ID NO:11) and containsan EcoRI restriction enzyme site behind the first 17 nucleotides of theTIMP-4 gene. The reverse primer has the sequence 5′CTAGTCTAGACTAGGGCTGGACGATGTCAA 3′ (SEQ ID NO:12) and contains a XbaIrestriction enzyme site and 24 nucleotides complementary to the 3′non-translated sequence of the TIMP-4 gene.

The amplified sequences were isolated from a 1% agarose gel using acommercially available kit (“Qiaquick”, Qiagen, Courtaboeuf, France),and then was EcoRI/Xba I subcloned in a transfer vector containing theCMV promoter. This transfer vector contains the Ad5 1-458 regionfollowed by the CMV enhancer/promoter and a chimeric intron generated bycombining the splice donor from the human beta-globin intron 1 and thesplice acceptor from the IgG intervening sequence obtained from pCIplasmid (Promega, Charbonnieres, France). A poly-linker containing,among others, the recognition sites for the restriction endonucleasesXbaI and EcoRI, was inserted upstream to the bovine growth hormonepolyadenylation site followed by the Ad5 3511-5788 region. This vectorcontains the ampicillin resistance gene.

The E1/E3-deleted adenoviral vector containing the gene encoding TIMP-4(named AdTG14854) was obtained by homologous recombination inEscherichia coli BJ (Chartier et al., J. Virol. 70(7): 4805-10 (1996)),between the TIMP-4 transfer vector (named pTG14846 for human andpTG14847 for rat; see FIGS. 3A and 3B) and the adenoviral DNA plasmid(named pTG6624; see FIG. 3F) linearized by ClaI. The adenoviral vectorcontaining human TIMP-4 (pTG14854; FIG. 3C) was deposited at theCollection Nationale de Cultures de Microorganismes, Institute Pasteur(25 Rue du Docteur Roux, F-74724 Paris Cedex 15, France), on Jul. 9,2001, and received deposit registration number CNCM 1-2696.

Virus propagation, purification and titration of infectious units (iu)by indirect immunofluorescence of the viral DNA binding protein werecarried out as described previously (Lusky et al., J. Virol.72(3):2022-32 (1998)). Purified virus was stored in viral storage buffer(1 M sucrose, 10 mM Tris-HCl [pH=8.5], 1 mM MgCl₂, 150 mM NaCl, 0.005%[vol/vol] Tween 80). Bacteria comprising DNA plasmid containing humanTIMP-4.

Cells and Culture Conditions:

Rat thoracic aorta smooth muscle cells were isolated from normal rats(ratAoSMCs) and from injured rats 15 days after balloon catheterdeendothelialization (ratIT5) by enzymatic digestion as previouslydescribed (Orlandi at al., Arterioscler. Thromb. 14(6):982-9 (1994)).Porcine coronary artery SMCs were isolated from normal pigs (pigCoSMCs)and from injured animals 15 days after stent placement (pigIT15) byenzymatic digestion as previously described (Christen et al., Circ. Res.85(1):99-107 (1999)). The human coronary artery SMCs were purchased fromClonetics (Walkersville, Md., USA). Rat and pig cells were cultured inDMEM containing 10% FCS (Life Technologies, Cergy-Pontoise, France).Human cells were cultured in SMGM2 medium containing 5% FCS(Bioproducts, Gagny, France).

Adenoviral Cell Infection:

SMCs were infected in suspension at an MOI corresponding to 80% ofinfected cells. Briefly, cells were trypsinized, centrifuged and thenresuspended in 2% FCS cell culture medium (5×10⁶ cells in 500 μl). Thevirus was added for a 30 min. incubation time at 37° C., 5% CO2. Cellswere rinsed in fresh medium and finally resuspended and plated in 10% or5% FCS corresponding medium.

Gelatin Zymography:

Recombinant human MMP2 was purchased from R&D System (Oxon, UK) and usedat 2.5 ng in the gel. Briefly, cell lysates (from 2×10⁵ cells) weremixed with Novex tris-Glycine Sample Buffer and let stand 10 minutes atroom temperature. Samples (20 μl) were then subjected to electrophoresison 10% Tris-Glycine gel with 0.1% gelatine incorporated as a substrate.Gels were washed in Novex Renaturing Buffer with gentle agitation for 30min. at room temperature. Renaturing Buffer is then decanted andreplaced with Developing Buffer for a 4 hour incubation at 37° C. Gelswere stained with Coomassie Blue R-250 for 30 min. Metalloproteinaseproduced clear areas of lysis in the gel.

Rat Carotid Artery Balloon Injury Model:

Adult male Wistar rats (body weight >400g) were used for experiments(Iffa-Credo). Anesthesia was induced with intraperitoneal injection ofKetamine (Imalgene, Rhône-Mérieux, Lyon, France) and Acepromazin(Vetranquil 0.5%, Sanofi, Libourne, France) in doses of 23.1 and 3.84mg/kg respectively. Animals were anticoagulated with intravenousinjection of 200 U/kg of human heparin (Choay, Sanofi Winthrop,Gentilly, France). The left common carotid artery was surgically exposedand an arteriotomy was made on the left external carotid artery.Deendothelialization was achieved by three passages of a 2F Fogartyballoon catheter (Baxter, Maurepas, France) filled with 0.2 ml air. A 1cm length segment of the carotid was isolated with microsurgical clampsand a 24-gauge catheter was introduced through the arteriotomy. Thesegment was flushed with 0.2 ml NaCl 0.9% and 50 μl of adenoviralsolution (2×10⁹ iu) was infused. The solution was allowed to dwell inthe carotid for 5 minutes during which the carotid segment remaineddistended. The solution was withdrawn, the external carotid artery wasligated and blood flow was reestablished through the common and theinternal carotid arteries. Rats were sacrificed at D14 post injury.After lethal pentobarbital injection and cannulation of the heart,vessels were perfused with 1x PBS solution and perfusion-fixed eitherwith 2% or 4% formaldehyde in PBS at normal blood pressure. Then,carotids were excised and treated for histological analyses.

For morphometric analysis, carotids were fixed in 4% formaldehyde andembedded in paraffin. Five μm sections were stained either withhematoxylin or with hematoxylin and eosin and the media and intimalarea, as well as medial and neointimal cell number were evaluated byimage analysis on 3 cross sections for each vessel (NIH Image software).

TIMP-4 mRNA Expression

Total RNA was extracted (RNA Now reagent, Ozyme, Montigny, France) at 24and 48 hours after AdTG14854 (CMV-human TIMP-4; see FIG. 3C) infectionof human and porcine CaSMCs, and AdTG14855 (CMV-rat TIMP-4; see FIG. 3D)infection of rat AoSMCs. The presence of the mRNA was detected byNorthern blot. Briefly, 10 μg of total RNA extracted from each of theabove cell populations were denatured and run on a 1% formaldehydeagarose gel and capillary blotted onto a Hybond nylon membraneovernight. RNA was fixed on the membrane by heating at 80° C. for 2hours. A random primer probe (Amersham Multi Prime Kit) was preparedfrom the SmaI-KpnI insert of the partial TIMP-4 nucleic acid sequenceand used to probe the blot by 3 hour hybridization in Amershamhybridization buffer containing 200 μg/ml denatured herring sperm DNA asa blocking agent. Washing was performed sequentially with 1× SSC-0.1%SDS (2×15 min.) and 0.1× SSC-0.1% SDS (1×10 min.). Results indicate thatthe exogeneous TIMP-4 mRNA is present in infected SMC of human andporcine coronary arteries and rat aorta. In all cell types, two majorbands were observed in agreement with published data (Gomez, EuropeanJournal of Cell Biology, 74:111 (1997)).

TIMP-4 Protein Expression

Total proteins were extracted from cells and culture supernatants 2days, 3 days, 5 days and 7 days after AdTG14854 infection of human andporcine CaSMCs, and AdTG14855 infection of rat AoSMCs. The presence ofTIMP-4 protein was detected by Western blot. Briefly, 150 μg ofextracted proteins were run on a 10% Nupage gel (Novex, Invitrogen,Groningen, the Netherlands), and then blotted on a nitrocellulosemembrane (Novex). The membrane was incubated in a blocking solution(PBS-2% milk) overnight before detection of the blotted antigen, usingan anti-TIMP-4 antibody (clone S720, Abcam, Cambridge, UK). Western blotdetection of the TIMP-4 protein indicates that TIMP-4 is present inlarge amounts in human coronary cell supernatants and in lower amountsin pig cell supernatants. In rat SMC supernatants, no protein wasdetected in IT15 and only a faint signal in ratAo. These resultsindicate either that the protein is not equally expressed by all celltypes or that the antibody does not recognize the rat TIMP-4. Byinfecting human cells with the rat TIMP-4 adenovirus and the rat cellswith the human TIMP-4 adenovirus, it was observed by western blot thatthe production was equivalent in the different cell types for a definedvector suggesting that the antibody is specific for the human protein.TIMP-4 activity

Staining of gelatin zymogram gels revealed a gelatin lysis activity (MMPactivity) with cell lysates corresponding to the Ad-null infectionwhereas, no areas of gel lysis were observed with cell lysate fromAd-TIMP-4 infected cells. Taken together with Western blot resultsshowing an equal amount of MMP2 in cells infected either with Ad-null orAd-TIMP-4 (data not shown), the zymogram data indicated that thedecrease of MMP activity was due to the TIMP-4 inhibitory effect.

Effects on Cell Proliferation

SMC proliferation was studied in SMCs infected in suspension at an MOIcorresponding to 80% of infected cells. The results indicate that on ratand pig cells, TIMP-4 is not able to significantly inhibit cell growth.On human cells, an inhibitory effect was observed which could be due toadenovirus toxicity. To confirm this hypothesis, lower MOIs of AdTIMP-4were used to infect human cells. The results are summarized in TableTable 1:

TABLE 1 MOI adenovirus MOI 0 MOI 1 MOI 10 MOI 50 MOI 100 300 AdTG6401100%  75% 75% 67% 44% 32.7% AdTG14854 100% 123% 84% 49% 30% 21.4%

An antiproliferative effect was observed at MOIs starting from MOI 50.At this and higher MOIs a toxic effect of the Adnull itself and a slightadditional toxicity of the TIMP-4 was observed. In conclusion, thesedata suggest that TIMP-4 has no growth inhibitory effect on rat, pig andhuman SMCs except at very high adenoviral load. These results are inagreement with published reports on the effects of other members of theTIMP family.

Effects on Cell Migration

The assay of migration in matrigel drops was used. TIMP-4 infected HumanCaSMCs were incorporated into 50 μl of matrigel. A non-selective MMPinhibitor (doxycycline) was added at different concentrations in the aimto inhibit most of the non-gelatinase (collagenase) activity. Inaddition, the medium was changed after 24 hours to remove soluble MMPs.Four days after seeding, an anti-migratory activity of TIMP-4 wasobserved. This effect is gelatinase-dependent since TIMP-4 anddoxycycline have cummulative effects. This experiment was repeated twicewith the same result.

TIMP-4 Inhibition of Neointimal Thickening in the Rat Injured CarotidModel

To examine the effect of Ad-TIMP-4 infection on neointimal formation, 12rats were infected after injury with 2×10⁹ IU of AdTG14855 or AdTG6401.The carotids of these animals were collected at 14 days after injury andboth neointimal and medial areas were measured (see Table 2). There wasa significant 74% reduction in neointimal area in Ad-TIMP-4 infectedvessels (1.04+/−0.32 mm2; p=0.00018; n=6) compared with Ad-null infectedvessels (5.03+/−1.66 mm²; n=6). No significant difference was seen inmedial area (5.86+/−0.32 versus 6.14+/−0.65 for AdTIMP-4 and Ad-Nullinfected vessels, respectively). As expected, the ratio of neointima tomedia showed a significant difference between Ad-TIMP-4 and Ad-Nullinfected vessels (0.18+/−0.05 versus 0.80+/−0.16; p=0.01). These resultsshow that adenovirus-mediated gene transfer of rat TIMP-4 to the ratcarotid artery immediately after injury, causes a significant decreasein neointima development.

TABLE 2 AdTG6401 (2 × 10⁹ iu) AdTG14855 (2 × 10⁹ iu) Lumen area 13.81 ±2.10  15.77 ± 1.84 (p = 0.11) Media area 6.14 ± 0.65  5.86 ± 0.32 (p =0.37) Intima area 5.03 ± 1.66  1.04 ± 0.32 (p = 0.00018) Neointima/media0.80 ± 0.16  0.18 ± 0.05 (p = 0.000012) Lumen perimeter 13.88 ± 0.88 15.25 ± 0.59 (p = 0.01)

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 8 <210> SEQ ID NO 1 <211> LENGTH: 675<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(672)<221> NAME/KEY: sig_peptide <222> LOCATION: (1)..(87)<221> NAME/KEY: mat_peptide <222> LOCATION: (88)..() <400> SEQUENCE: 1atg cct ggg agc cct cgg ccc gcg cca agc tg#g gtg ctg ttg ctg cgg       48Met Pro Gly Ser Pro Arg Pro Ala Pro Ser Tr #p Val Leu Leu Leu Arg                -25   #               -20   #               -15ctg ctg gcg ttg ctg cgg ccc ccg ggg ctg gg#t gag gca tgc agc tgc       96Leu Leu Ala Leu Leu Arg Pro Pro Gly Leu Gl #y Glu Ala Cys Ser Cys            -10       #           -5        #       -1  1gcc ccg gcg cac cct cag cag cac atc tgc ca#c tcg gca ctt gtg att      144Ala Pro Ala His Pro Gln Gln His Ile Cys Hi #s Ser Ala Leu Val Ile    5               #     10              #     15cgg gcc aaa atc tcc agt gag aag gta gtt cc#g gcc agt gca gac cct      192Arg Ala Lys Ile Ser Ser Glu Lys Val Val Pr #o Ala Ser Ala Asp Pro20                   #25                   #30                   #35gct gac act gaa aaa atg ctc cgg tat gaa at#c aaa cag ata aag atg      240Ala Asp Thr Glu Lys Met Leu Arg Tyr Glu Il #e Lys Gln Ile Lys Met                40   #                45   #                50ttc aaa ggg ttt gag aaa gtc aag gat gtt ca#g tat atc tat acg cct      288Phe Lys Gly Phe Glu Lys Val Lys Asp Val Gl #n Tyr Ile Tyr Thr Pro            55       #            60       #            65ttt gac tct tcc ctc tgt ggt gtg aaa cta ga#a gcc aac agc cag aag      336Phe Asp Ser Ser Leu Cys Gly Val Lys Leu Gl #u Ala Asn Ser Gln Lys        70           #        75           #        80cag tat ctc ttg act ggt cag gtc ctc agt ga#t gga aaa gtc ttc atc      384Gln Tyr Leu Leu Thr Gly Gln Val Leu Ser As #p Gly Lys Val Phe Ile    85               #    90               #    95cat ctg tgc aac tac atc gag ccc tgg gag ga#c ctg tcc ttg gtg cag      432His Leu Cys Asn Tyr Ile Glu Pro Trp Glu As #p Leu Ser Leu Val Gln100                 1 #05                 1 #10                 1 #15agg gaa agt ctg aat cat cac tac cat ctg aa#c tgt ggc tgc caa atc      480Arg Glu Ser Leu Asn His His Tyr His Leu As #n Cys Gly Cys Gln Ile                120   #               125   #               130acc acc tgc tac aca gta ccc tgt acc atc tc#g gcc cct aac gag tgc      528Thr Thr Cys Tyr Thr Val Pro Cys Thr Ile Se #r Ala Pro Asn Glu Cys            135       #           140       #           145ctc tgg aca gac tgg ctg ttg gaa cga aag ct#c tat ggt tac cag gct      576Leu Trp Thr Asp Trp Leu Leu Glu Arg Lys Le #u Tyr Gly Tyr Gln Ala        150           #       155           #       160cag cat tat gtc tgt atg aag cat gtt gac gg#c acc tgc agc tgg tac      624Gln His Tyr Val Cys Met Lys His Val Asp Gl #y Thr Cys Ser Trp Tyr    165               #   170               #   175cgg ggc cac ctg cct ctc agg aag gag ttt gt#t gac atc gtt cag ccc      672Arg Gly His Leu Pro Leu Arg Lys Glu Phe Va #l Asp Ile Val Gln Pro180                 1 #85                 1 #90                 1 #95tag                   #                   #                  #            675 <210> SEQ ID NO 2 <211> LENGTH: 224 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 2Met Pro Gly Ser Pro Arg Pro Ala Pro Ser Tr #p Val Leu Leu Leu Arg                -25   #               -20   #               -15Leu Leu Ala Leu Leu Arg Pro Pro Gly Leu Gl #y Glu Ala Cys Ser Cys            -10       #           -5        #       -1  1Ala Pro Ala His Pro Gln Gln His Ile Cys Hi #s Ser Ala Leu Val Ile    5               #     10              #     15Arg Ala Lys Ile Ser Ser Glu Lys Val Val Pr #o Ala Ser Ala Asp Pro20                   #25                   #30                   #35Ala Asp Thr Glu Lys Met Leu Arg Tyr Glu Il #e Lys Gln Ile Lys Met                40   #                45   #                50Phe Lys Gly Phe Glu Lys Val Lys Asp Val Gl #n Tyr Ile Tyr Thr Pro            55       #            60       #            65Phe Asp Ser Ser Leu Cys Gly Val Lys Leu Gl #u Ala Asn Ser Gln Lys        70           #        75           #        80Gln Tyr Leu Leu Thr Gly Gln Val Leu Ser As #p Gly Lys Val Phe Ile    85               #    90               #    95His Leu Cys Asn Tyr Ile Glu Pro Trp Glu As #p Leu Ser Leu Val Gln100                 1 #05                 1 #10                 1 #15Arg Glu Ser Leu Asn His His Tyr His Leu As #n Cys Gly Cys Gln Ile                120   #               125   #               130Thr Thr Cys Tyr Thr Val Pro Cys Thr Ile Se #r Ala Pro Asn Glu Cys            135       #           140       #           145Leu Trp Thr Asp Trp Leu Leu Glu Arg Lys Le #u Tyr Gly Tyr Gln Ala        150           #       155           #       160Gln His Tyr Val Cys Met Lys His Val Asp Gl #y Thr Cys Ser Trp Tyr    165               #   170               #   175Arg Gly His Leu Pro Leu Arg Lys Glu Phe Va #l Asp Ile Val Gln Pro180                 1 #85                 1 #90                 1 #95<210> SEQ ID NO 3 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: 5′ TIMP-4 primer with  #BamH1site<400> SEQUENCE: 3 gccagaggat cctgcagctg cgccccggcg cac       #                   #         33 <210> SEQ ID NO 4 <211> LENGTH: 33<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: 3′ TIMP-4 primer with  #XbaI site<400> SEQUENCE: 4 cggcttctag aactagggct gaacgatgtc aac       #                   #         33 <210> SEQ ID NO 5 <211> LENGTH: 39<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: 5′ TIMP-4 primer with  #BamHI site<400> SEQUENCE: 5 gccagaggat ccgccaccat gcctgggagc cctcggccc      #                   #    39 <210> SEQ ID NO 6 <211> LENGTH: 60<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: 3′ TIMP-4 primer with  #XbaI site<400> SEQUENCE: 6cggcttctag aatcaagcgt agtctgggac gtcgtatggg tagggctgaa cg#atgtcaac     60 <210> SEQ ID NO 7 <211> LENGTH: 33 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: 5′ TIMP-4 primer with  #BamHI site<400> SEQUENCE: 7 gccagaggat ccatgcctgg gagccctcgg ccc       #                   #         33 <210> SEQ ID NO 8 <211> LENGTH: 33<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: 3′ TIMP-4 primer with  #XbaI site<400> SEQUENCE: 8 cggcttctag aactagggct gaacgatgtc aac       #                   #         33

What is claimed is:
 1. An isolated protein consisting of at least 30contiguous amino acids of SEQ ID NO:2, wherein said protein inhibitsmetalloproteinase activity.
 2. The protein of claim 1 which consists ofat least 50 contiguous amino acids.
 3. The protein of claim 1 fused to aheterologous polypeptide.
 4. The protein of claim 1 which isglycosylated.
 5. A composition comprising the protein of claim 1 in apharmaceutically acceptable carrier.
 6. A TIMP-4 protein produced by amethod comprising: (a) culturing a host cell under conditions suitableto produce the protein of claim 1; and (b) recovering said TIMP-4protein from the host cell culture.
 7. An isolated protein consisting ofat least 30 contiguous amino acids of the full-length polypeptideencoded by the cDNA contained in ATCC Deposit No. 75946, wherein saidprotein inhibits metalloproteinase activity.
 8. The protein of claim 7which consists of at least 50 contiguous amino acids.
 9. The protein ofclaim 7 fused to a heterologous polypeptide.
 10. The protein of claim 7which is glycosylated.
 11. A composition comprising the protein of claim7 in a pharmaceutically acceptable carrier.
 12. A TIMP-4 proteinproduced by a method comprising: (a) culturing a host cell underconditions suitable to produce the protein of claim 7; and (b)recovering said TIMP-4 protein from the host cell culture.
 13. Anisolated protein comprising a first amino acid sequence 90% or moreidentical to a second amino acid sequence selected from the groupconsisting of: (a) amino acid residues −29 to 195 of SEQ ID NO:2; and(b) amino acid residues 1 to 195 of SEQ ID NO:2; wherein said proteininhibits metalloproteinase activity.
 14. The protein of claim 13 whichcomprises a first amino acid sequence 90% or more identical to (a). 15.The protein of claim 13 which comprises a first amino acid sequence 90%or more identical to (b).
 16. The protein of claim 13 which comprises afirst amino acid sequence 95% or more identical to (a).
 17. The proteinof claim 13 which comprises a first amino acid sequence 95% or moreidentical to (b).
 18. The protein of claim 13 fused to a heterologouspolypeptide.
 19. The protein of claim 13 which is glycosylated.
 20. Acomposition comprising the protein of claim 13 in a pharmaceuticallyacceptable carrier.
 21. A TIMP-4 protein produced by a methodcomprising: (a) culturing a host cell under conditions suitable toproduce the protein of claim 13; and (b) recovering said TIMP-4 proteinfrom the host cell culture.
 22. An isolated protein comprising a firstamino acid sequence 90% or more identical to a second amino acidsequence selected from the group consisting of: (a) the amino acidsequence of the full-length polypeptide encoded by the cDNA contained inATCC Deposit No.75946; and (b) the amino acid sequence of the maturepolypeptide encoded by the cDNA contained in ATCC Deposit No. 75946;wherein said protein inhibits metalloproteinase activity.
 23. Theprotein of claim 22 which comprises a first amino acid sequence 90% ormore identical to (a).
 24. The protein of claim 22 which comprises afirst amino acid sequence 90% or more identical to (b).
 25. The proteinof claim 22 which comprises a first amino acid sequence 95% or moreidentical to (a).
 26. The protein of claim 22 which comprises a firstamino acid sequence 95% or more identical to (b).
 27. The protein ofclaim 22 fused to a heterologous polypeptide.
 28. The protein of claim22, wherein said protein is glycosylated.
 29. A composition comprisingthe protein of claim 22 in a pharmaceutically acceptable carrier.
 30. ATIMP-4 protein produced by a method comprising: (a) culturing a hostcell under conditions suitable to produce the protein of claim 22; and(b) recovering said TIMP-4 protein from the host cell culture.